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WO2001013960A1 - Procedes d'administration de genes in vivo a des sites de deterioration de cartilage - Google Patents

Procedes d'administration de genes in vivo a des sites de deterioration de cartilage Download PDF

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Publication number
WO2001013960A1
WO2001013960A1 PCT/US2000/022801 US0022801W WO0113960A1 WO 2001013960 A1 WO2001013960 A1 WO 2001013960A1 US 0022801 W US0022801 W US 0022801W WO 0113960 A1 WO0113960 A1 WO 0113960A1
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Prior art keywords
polynucleotide
medicament
subchondral
joint
perforation
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PCT/US2000/022801
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English (en)
Inventor
Steven C. Ghivizzani
Christopher H. Evans
Paul D. Robbins
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University Of Pittsburgh Of The Commonwealth System Of Higher Education
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Priority to AU69193/00A priority Critical patent/AU6919300A/en
Publication of WO2001013960A1 publication Critical patent/WO2001013960A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2006—IL-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/30—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • 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
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein

Definitions

  • the present invention relates to improved methods for transferring genes to cells in vivo.
  • it relates to the use of gene therapy techniques to transfect subchondral cells in animal joints.
  • Such methods can be used to introduce marker genes into the cells, or to introduce genes into the cells that promote chondro genesis in the joint.
  • chondrocytes the cells within the cartilage that could potentially promote cartilage growth, called chondrocytes, are shut off from entities likely to stimulate healing, such as circulating factors, hormones, or cells.
  • the chondrocytes are embedded within a dense matrix, and are thus unable to migrate or traffic, thus preventing their travel to sites of damage.
  • partial-thickness defect refers to cartilage damage that does not extend to the subchondral bone. Partial-thickness defects are especially resistant to healing, and the chondrocytes in such cases exhibit almost no response to the defect. Full thickness defects, which extend into the subchondral bone, evoke a greater response, but nevertheless fail to produce a satisfactory repair tissue. Full thickness defects often degenerate into osteoarthritis, and in many cases ultimately require the surgical replacement of the joint. Clearly, improved methods for treating both partial thickness and full thickness defects are needed.
  • chondrocytes which reside in the cartilage and are responsible for maintaining the extracellular matrix of the cartilage
  • the extracellular mat ⁇ x provides the mechanical properties of the cartilage, such as its shock absorbing, load bearing, and protective properties
  • Chondrocytes are capable of both producing and degrading the extracellular matnx, and are thought to, m a healthy joint, maintain a proper balance between mat ⁇ x production and degradation so as to maintain normal cartilage It is thought that some diseases, such as osteoarthritis, result from the loss of this balance in chondrocyte metabolism, resulting in an overall net loss of cartilage
  • Some efficacy m treating cartilage injury is the administration of cultured chondrocyte cells to joints with damaged cartilage
  • B ⁇ ttberg et al (New Eng J Med 331 889 (1994)) introduced cultured, autologous chondrocytes into defects of the articular cartilage While this treatment showed some positive results, the success was va ⁇ able and limited
  • chondrocytes have been cultured and transfected in vitro, and the transfected cells transplanted into a joint with a cartilage defect (see, e.g., Kang et al., (1997) Osteoarthritis and Cartilage 5: 139- 143; Glorioso et al. , U.S. Patent Application Serial No. 08/466,932, filed April 6, 1995; Baragi et al, (1997) Osteoarthritis and Cartilage 5:275-282; Doherty et al., (1998) Osteoarthritis and Cartilage, 6: 153-160; Baragi et al , (1995) J. Clin. Invest. 96: 1995).
  • chondroprogenitor cells including the mesenchymal stem cells.
  • mesenchymal stem cells are located within the bone marrow (Wakitani, S. et al, J. Bone Jt Surg 76A:579 (1994)), periosteum (Oleksyszyn, J. et al, Inflamm Res 45:464 (1996)) and, perhaps, elsewhere (Hunziker, E.B. et al, J Bone Jt Surg 78A:721 (1996); Iwata, H. et al., Clin Orthop Rel Res 296:295 (1993)).
  • Mesenchymal stem cells can differentiate into any of a number of cell types, including cartilage, bone, and tendon.
  • the inaccessibility of chondroprogenitor and mesenchymal stem cells has limited therapeutic methods involving such cells to ex vivo approaches (see, e.g., Glorioso et al, WO9639196; Allay, J.A. et al, Human Gene Ther 8: 1417 (1997); Balk, M.L. et al, Bone 21 :7 (1997)).
  • these cell types have been isolated from bone marrow, grown in culture, and introduced into damaged joints as a potential strategy for joint repair.
  • the present invention provides methods of expressing heterologous proteins in subchondral cells in vivo.
  • the present methods involve exposing subchondral cells in vivo and transfectmg tne cells with a polynucteotide encoding the heterologous protein, whereby the polynucleotide is expressed
  • the present methods involve providing a heterologous protein m a subchondral cell m a joint of a mammal by creating a perforation m a subchondral bone of the joint, and introducing a polynucleotide encoding the heterologous protein into the perforation, whereby a subchondral cell, e g , a chondroprogenitor or mesenchymal stem cell that is exposed by or localized to the perforation internalizes the polynucleotide and expresses the heterologous protein
  • the perforation can be produced by a number of means, e g , d ⁇ ll g, microfracture, or abrasion
  • the perforation is produced by one of these means arthroscopically
  • the polynucleotide is introduced into the perforation in the subchondral bone using an artificial mat ⁇ x, for example collagen
  • the heterologous protein is a chondrogenesis- promoting polypeptide
  • the chondrogenesis- promotmg polypeptide is a growth factor (e g , TGF- ⁇ ), a cartilage-de ⁇ ved morphogenetic factor, a cytokine inhibitor (e g , an ⁇ nterleukm-1 receptor antagonist), or a protemase inhibitor
  • the polynucleotide can be introduced using a va ⁇ ety of means, for example using an adenoviral vector, a retroviral vector, formulated plasmid DNA, naked DNA, an adeno-associated viral vector, a herpes simplex viral vector, or other non-viral DNA formulations
  • the mammal has arth ⁇ tis, osteoarth ⁇ tis, osteochond ⁇ tis dissecans, avascular necrosis, or has undergone an injury to the joint
  • Pharmaceutical formulations and kits for practicing the present methods are also provided
  • expression cassette refers to a nucleic acid sequence to be introduced into a cell and contains the nucleic acid sequence to be transcribed and a promoter to direct the transcription.
  • the promoter can either be homologous, i.e., occurring naturally to direct the expression of the desired nucleic acid or heterologous, i.e., occurring naturally to direct the expression of a nucleic acid derived from a gene other than the desired nucleic acid. Fusion genes with heterologous promoter sequences are desirable, e.g., for regulating expression of encoded proteins.
  • the promoter may constitutively bind transcription factors and RNA Polymerase II.
  • a heterologous promoter may be desirable because it has sequences that bind transcription factors the naturally occurring promoter lacks.
  • an expression cassette preferably contains termination sequences and a poly A+ signal for, e.g. , mRNA stabilization.
  • sequence comparison algorithms refers to the residues in the two sequences that are the same when aligned for maximum correspondence, as measured using one of the following "sequence comparison algorithms.”
  • substantially identical in the context of two polypeptides refers to the residues in the two sequences that have at least 60% identity when aligned for maximum correspondence over a domain of the protein.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol Biol 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol Evol 35:351-360 (1987). The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153 (1989). The program can align up to 300 sequences of a maximum length of 5,000. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster can then be aligned to the next most related sequence or cluster of aligned sequences.
  • Two clusters of sequences can be aligned by a simple extension of the pairwise alignment of two individual sequences.
  • the final alignment is achieved by a series of progressive, pairwise alignments.
  • the program can also be used to plot a dendrogram or tree representation of clustering relationships. The program is run by designating specific sequences and their amino acid coordinates for regions of sequence comparison.
  • HSPs high scoring sequence pairs
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST program uses as defaults a wordlength (W) of 1 1, the BLOSUM62 scoring matrix (see H ⁇ nikoff & Henikoff, Proc. Nad. Acad. Sci. USA 89: 10915 (1989)) alignments (B) of 50, expectation (E) of 10, M'5, N'-4, and a comparison of both strands.
  • the BLAST algorithm performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat ' Acad. Sci. USA 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • a nucleic acid is considered similar to an ribonuclease nucleic acid if the smallest sum probability in a comparison of the test nucleic acid to an ribonuclease nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • the test nucleic acid encodes a ribonuclease polypeptide
  • it is considered similar to a specified ribonuclease nucleic acid if the comparison results in a smallest sum probability of less than about 0.5, and more preferably less than about 0.2.
  • Another indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with the second polypeptide.
  • a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution.
  • isolated refers to material that is substantially or essentially free from components which normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A polypeptide that is the predominant species present in a preparation is substantially purified.
  • purified denotes that a polypeptide gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the polypeptide is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
  • nucleic acid refers to any deoxyribonucleotide or ribonucleotide sequence in, e.g., single-stranded, double-stranded or triplex form.
  • the terms encompass nucleic acids, e.g., oligonucleotides, containing known naturally occurring nucleotides, analogues of natural nucleotides, and mixtures thereof.
  • the term also encompasses nucleic-acid-like structures with synthetic backbones.
  • DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methyl-phosphonate , amino phosphonate, phosphor- amidate, alkyl phosphotri ester, sulfamate, 3'-thioacetal, methylene (methylimino), 3'-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds.
  • PNAs contain non-ionic backbones, such as N-(2- aminoethyl) glycine units. Phosphorothioate linkages are described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144: 189-197.
  • nucleic acids can comprise any of a number of nucleotide analogs, i.e., nucleotides modified to include fluorescent moieties, radioactive isotopes, biotin, digoxigenin, 2,4-Dinitrophenyl (DNP), 5' Bromo-2'-deoxyUridine (BrdU), chemically reactive side chains, alternative bases such as inosine, uridine, or others.
  • nucleotide analogs i.e., nucleotides modified to include fluorescent moieties, radioactive isotopes, biotin, digoxigenin, 2,4-Dinitrophenyl (DNP), 5' Bromo-2'-deoxyUridine (BrdU), chemically reactive side chains, alternative bases such as inosine, uridine, or others.
  • Nucleic acids can be of any length, including short polymers such as oligonucleotides, as well as longer sequences such as amplification fragments, restriction fragments, plasmids, and other vectors.
  • the "nucleic acids” or “polynucleotides” can be made through any standard method, such as by chemical or other in vitro synthesis, by amplification reactions, or by isolation from cells, and can include naturally-occurring as well as recombinant sequences.
  • the term "pharmaceutically acceptable carrier” includes any suitable pharmaceutical excipient, including, e.g., water, saline, phosphate buffered saline, Hank's solution, Ringer's solution, dextrose/saline, glucose, lactose, or sucrose solutions, starch, cellulose, talc, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, dried skim milk, glycerol, propylene glycol, ethanol, and the like.
  • suitable pharmaceutical excipient including, e.g., water, saline, phosphate buffered saline, Hank's solution, Ringer's solution, dextrose/saline, glucose, lactose, or sucrose solutions, starch, cellulose, talc, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate
  • Recombinant when used with reference to a protein indicates that a cell expresses a peptide or protein encoded by a nucleic acid whose origin is exogenous to the cell.
  • Recombinant cells can express genes that are not found within the native (non- recombinan ) form of the cell.
  • Recombinant cells can also express genes found in the native form of the cell wherein the genes are re-introduced into the cell by artificial means, for example under the control of a heterologous promoter or using a plasmid or viral vector.
  • the phrase ' " reduces the symptoms of or "ameliorating” or '"ameliorate” refers to any indicia of success in the treatment of a pathology or condition, including any objective or subjective parameter such as abatement, remission or diminishing of symptoms or an improvement in a patient's physical or mental well-being. Amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination and/or a psychiatric evaluation.
  • the phrase “specifically (or selectively) binds” refers to a binding reaction that is determinative of the presence of a polypeptide in a heterogeneous population of polypeptides and other compounds. Thus, under designated binding conditions, the specified polypeptides bind to a particular compound at least two times the background and do not substantially bind in a significant amount to other compounds present in the sample.
  • Transfection means the delivery of exogenous nucleic acid molecules to a cell, either in vivo or in vitro, whereby the nucleic acid is taken up by the cell and is functional within the cell.
  • a cell that has taken up the exogenous nucleic acid is refe ⁇ ed to as a "host cell,” “target cell,” “transduced cell,” or “transfected cell.”
  • a nucleic acid is functional within a host cell when it is capable of functioning as intended.
  • the exogenous nucleic acid will comprise an expression cassette which includes DNA coding for a gene of interest, with appropriate regulatory elements, which will have the intended function if the DNA is transcribed and translated, thereby causing the host cell to produce the peptide or protein encoded therein.
  • DNA may encode a protein lacking in the transfected cell, or produced in insufficient quantity or less active form, or secreted, where it may have an effect on cells other than the transfected cell.
  • Other examples of exogenous nucleic acid to be delivered include, e.g., antisense oligonucleotides, mRNA ribozymes, or DNA encoding antisense RNA or ribozymes.
  • Nucleic acids of interest also include DNA coding for a cellular factor which, when expressed, activates the expression of an endogenous gene.
  • a cell can be "transfected” or “transduced” using any of the methods provided herein, including those using viral based vectors, naked DNA, or DNA formulations such as liposomal formulations.
  • a "polypeptide,” or “protein” is a polymer comprising monomers that are interconnected by peptide linkages. The monomers are most often the twenty naturally occurring amino acids (see. e.g. Alberts et a!, (1989) Molecular Biology of the Cell, Garland Publishing, Inc.), but may also include amino acid analogs or other compounds that can be linked using peptide chemistry.
  • a “heterologous protein” refers to any polypeptide that has been introduced into a cell or that is expressed from a polynucleotide that has been introduced into a cell.
  • a “heterologous protein” can comprise a different sequence than a naturally expressed protein, can have the same sequence but be expressed at a different level or with altered timing or regulation than an endogenous protein, or can represent a polynucleotide identical to an endogenous gene that has been introduced, e.g., to replace a defective endogenous gene or to increase the natural level of expression of a protein.
  • Cartilage refers to connective tissue comprising collagen and proteoglycan, particularly chondroitin sulphate. Cartilage is typically produced by chondrocytes that come to lie in small lacunae surrounded by the matrix they have secreted.
  • intra-articular refers to the interior of a joint, e.g., knee, elbow, shoulder, ankle, wrist, etc.
  • an intra-articular injection is an injection into the space between the bones of a joint.
  • intra-articular refers to the space between the femur and the tibia, behind and surrounding the patella.
  • an “artificial matrix,” or “implanted matrix” refers to any substance in which molecules, cells, or other materials can be embedded. Commonly, an “artificial” or “implanted” “matrix” will comprise a regular framework within with such items can be embedded, and will be composed of a biological or other polymer, e.g., collagen. “Subchondral bone” refers to any bone located beneath cartilage.
  • Subchondral cell refers to any cell, such as a chondroprogenitor or mesenchymal stem cell, that is exposed by or localized to a perforation introduced or existing in a subchondral bone.
  • the cell can be directly exposed to the exterior of the animal, or can simply be placed in fluidic or other contact with the exterior, thereby allowing the application of material, e.g., a polynucleotide formulation, to the cell.
  • a “chondroprogenitor cell” is any cell that is capable of promoting chondrogenesis, e.g., by differentiating into a chondrocyte, by promoting cartilage synthesis, by inhibiting cartilage degradation, and the like.
  • “Mesenchymal stem cells” refer to cells, present within the bone marrow, that can differentiate to form cells associated with any of a number of tissue types, such as cartilage, bone, fat, muscle, and other tissues.
  • arthritis refers to any of a number of inflammatory or degenerative conditions that affects joints. "Arthritis” can result from any of a number of causes, including infection, autoimmune disorders, or injury.
  • osteoarthritis refers to a noninflammatory degenerative joint disease characterized by degeneration of the articular cartilage, hypertrophy of bone at the margins and changes in the synovial membrane. Also known as degenerative joint disease, osteoarthritis is the most common form of arthritis.
  • Cartilage injury refers to any damage or lesion in a cartilage tissue. In cases of articular cartilage, injuries can occur through any of a number of means, including from direct trauma resulting, e.g., in damage from rotational forces, shearing injuries, secondary effects of surgery, and the like, or from any of a number of degenerative or inflammatory injuries such as osteoarthritis.
  • a "cartilage injury” can occur in any part of the cartilage, such as medial, lateral, etc., and can refer to any type of lesion or damage, such as fibrillation, discoloration, softness, cracks, tears, craters, etc.
  • a “cartilage injury” can be “full-thickness,” meaning that the injury extends through the cartilage and into the subchondral bone, or can be “partial thickness,” meaning that the injury does not extend completely through the cartilage into the subchondral bone.
  • a "perforation" in a bone refers to any hole, breach, fracture, aperture, piercing, or any other opening that places cells within the bone, blood stream, or bone marrow, e.g. chondroprogenitor cells, into contiguous contact with the exterior of the bone.
  • a "perforation" in a subchondral bone will expose the chondroprogenitor cells within the marrow of the bone, thereby allowing the application of molecules, e.g., nucleic acids, to such cells.
  • a “perforation” can be introduced into a bone by any of a number of mechanisms, including, but not limited to, drilling, abrasion, microfracturing, and piercing. It will be appreciated that a perforation can be created expressly for the practice of the present invention, or can be pre-existing, e.g., the result of a previous injury or degenerative disease or disorder.
  • a “chondrogenesis-promoting polypeptide,” or a “chondrogenic polypeptide,” is any polypeptide that causes a net increase in cartilage synthesis, chondrocyte levels, or any property typical of healthy cartilage or articulations, e.g., the resilience, shock absorbing ability, etc. of the cartilage.
  • Such factors can include growth factors that stimulate growth and/or proliferation of chondroprogenitor cells or chondrocytes, factors that promote the differentiation of chondroprogenitor cells into chondrocytes, e.g., cartilage-derived morphogenetic factors, cytokine inhibitors, e.g., interleukin-1 receptor antagonists, etc.
  • “Localizing" a polynucleotide to a joint refers to the introduction of the polynucleotide into the joint in such a way that the local concentration of the polynucleotide remains at a significant level for an extended period of time, i.e., the polynucleotide does not immediately diffuse away from the site of administration.
  • “Localizing" a polynucleotide to a joint facilitates the transfection of cells within the joint after the initial moment of administration, e.g., cells migrating into the joint subsequent to administration of the polynucleotide can be readily transfected.
  • Figure 1 shows luciferase expression following implantation of adenoviral vectors into cartilage defects in joints.
  • Figure 2 shows luciferase expression following non-viral delivery of the luciferase gene into cartilage defects in joints.
  • Figure 3 shows production of insulin-like growth factor 1 (IGF-1) by transduced chondrocytes and its effects on matrix synthesis.
  • Figure 3 A shows IGF-1 concentrations in conditioned media following transduction with the IGF-1 gene or addition of IGF-1 protein.
  • Figure 3B shows the effects of IGF-1 protein and gene on the synthesis of proteoglycans by chondrocytes.
  • Figure 3C shows the effects of IGF-1 protein and gene on the synthesis of collagen and noncollagenous proteins by chondrocytes.
  • Figure 4 shows the additive effects of growth factor (insulin-like growth factor 1 [IGF-1] and transforming growth factor ⁇ 1 [TGF ⁇ l]) gene combinations on proteoglycan synthesis by chondrocytes.
  • Figure 5 shows the effects of growth factor (transforming growth factor ⁇ l [TGF ⁇ l] and insulin-like growth factor 1 [IGF-1]) genes on proteoglycan synthesis in the presence of interleukin-1 (IL-1).
  • IL-1 interleukin-1
  • the present invention provides methods of transfecting subchondral cells in vivo. Using these methods, subchondral cells within a bone or bone marrow, or cells within the bloodstream, are exposed by introducing a perforation into a subchondral bone in a joint. These exposed subchondral cells are then transfected with a polynucleotide encoding a protein of interest, leading to expression of the polynucleotide in vivo. Such methods are useful for numerous applications, including providing a marker for subchondral cells in vivo, and promoting chondrogenesis by expressing a chondrogenesis-promoting polynucleotide, e.g., a growth factor, in the cells.
  • a chondrogenesis-promoting polynucleotide e.g., a growth factor
  • the methods entail multiple steps, including providing a perforation into a subchondral bone by any method, e.g., drilling, piercing, abrasion, or microfracturing the bone, thereby exposing the cells. Such methods are preferably performed arthroscopically. Once a perforation is obtained, the exposed cells are preferably contacted with the polynucleotide by formulating the polynucleotide in an artificial matrix, e.g. a collagen matrix, and introducing the matrix-polynucleotide formulation into the perforation.
  • an artificial matrix e.g. a collagen matrix
  • the nucleic acid will typically encode a marker gene or a gene that promotes the growth-, proliferation-, and/or chondrogenic differentiation of subchondral cells, or a gene that promotes the in vivo synthesis of cartilage components, e.g., collagen, thereby promoting cartilage growth in a joint.
  • the present methods can be used to mark subchondral cells in a joint, thereby providing a method for monitoring the growth, proliferation, survival, localization, etc., of these cells in vivo.
  • Such methods are useful in any animal, including humans and animal models, e.g., to monitor the efficacy of a treatment for a cartilage defect, such as a method aimed to promote the growth of cells, including chondroprogenitor or mesenchymal stem cells, in vivo.
  • a method aimed to promote the growth of cells including chondroprogenitor or mesenchymal stem cells, in vivo.
  • the ability to monitor subchondral cells in vivo is also useful to monitor the progression of a disease or condition, e.g., osteoarthritis, over time. This ability provides important information regarding the nature and severity of a disease that can be used to inform decisions regarding optimal treatment for the disease.
  • the present methods can also be used to introduce chondrogenesis-promoting nucleotides into subchondral cells in a joint, e.g., a joint with a cartilage injury, of an animal such as a human or an animal model.
  • a gene that promotes chondrogenic differentiation of subchondral cells, or of the growth and/or proliferation of subchondral cells can be used.
  • genes that promote the production of cartilage components, e.g., collagen, or that inhibit the degradation of cartilage components can be used. Such methods are useful to promote cartilage growth in joints with cartilage defects or injury, and are thus useful as methods of treatment for such conditions.
  • Any of the herein-described methods can be performed on any animal, preferably on mammals including, but not limited to, humans.
  • the methods of the invention involve delivery of genes to cells exposed by a perforation in a subchondral bone in a joint.
  • the methods also typically involve the use of techniques to introduce at least one perforation in the subchondral bone to expose cells present in the bone to the vectors of the invention.
  • cells can be exposed by any method that penetrates the subchondral bone, allowing bleeding and/or entry of bone marrow into the lesion. Methods with which to do this include drilling, abrasion, and microfracture, e.g., using an arthroscopic awl to make one or more perforations in the subchondral bone. All of these methods are preferably performed arthroscopically.
  • adenoviral vector mediated gene delivery see, e.g., Chen et al. (1994) Proc. Nat 'I. Acad. Sci. USA 91 : 3054-3057; Tong et al. (1996) Gynecol Oncol. 61 : 175-179; Clayman et al (1995) Cancer Res. 5: 1-6; O'Malley et al. (1995) Cancer Res.
  • retroviral vectors see, e.g., Marx et al Hum Gene Ther 1999 May 1;10(7):1163-73; Mason et al, Gene Ther 1998 Aug;5(8): 1098-104.
  • replication-defective retroviral vectors harboring a therapeutic polynucleotide sequence as part of the retroviral genome have also been used, particularly with regard to simple MuLV vectors. See, e.g., Miller et al (1990) Mol. Cell Biol 10:4239 (1990); Kolberg (1992) J. NIH Res. 4:43, and Cornetta et al. Hum. Gene Ther. 2:215 (1991)).
  • retroviral vectors include lentiviruses (Klimatcheva et al, (1999) Front Biosci 4:D481-96).
  • viral vectors that can be used in the present invention include vectors derived from adeno-associated viruses (Bueler (1999) Biol Chem 380(6):613-22; Robbins and Ghivizzani (1998) Pharmacol Ther 80(l):35-47), herpes simplex viruses (Krisky et al, (1998) Gene Ther 5(1 1):1517-30 ), and others.
  • Plasmid vectors are typically delivered as "naked” DNA or combined with various transfection-facilitating agents. Numerous studies have demonstrated the direct administration of naked DNA, e.g., plasmid DNA, to cells in vivo (see, e.g., Wolff, Neuromuscul Disord 1997 Jul;7(5):314-8, Nomura et al, Gene Ther. 1999 Jan;6(l):121- 9). For certain applications it is possible to coat the DNA onto small particles and project genes into cells using a device known as a gene gun.
  • Plasmid DNA can also be combined with any of a number of transfection- facilitating agents. When combined with any such agents, the plasmid DNA is referred to herein as "formulated plasmid DNA.”
  • the most commonly used transfection facilitating agents for plasmid DNA in vivo has been charged and/or neutral lipids (Debs and Zhu (1993) WO 93/24640 and U.S. Pat. No. 5,641,662; Debs U.S. Pat. No. 5,756,353; Debs and Zhu Published EP Appl. No. 93903386; Mannino and Gould-Fogerite (1988) BioTechniques 6(7): 682-691; Rose U.S. Pat No.
  • Immunoliposomes have been described as carriers of exogenous polynucleotides (Wang and Huang, 1987, Proc. Natl. Acad. Sci. U.S.A. 84:7851 ; Trubetskoy et al., 1992, Biochem. Biophys. Acta 1131 :311) and may have improved cell type specificity as compared to liposomes by virtue of the inclusion of specific antibodies which presumably bind to surface antigens on specific cell types. Behr et al, 1989, Proc. Natl Acad. Sci. U.S.A.
  • Lipopolyamine as a reagent to mediate transfection itself, without the necessity of any additional phospholipid to form liposomes.
  • Lipid earners usually contain a cationic lipid and a neutral lipid.
  • Most in vivo transfection protocols involve forming liposomes made up of a mixture of cationic and neutral lipid and complexing the mixture with a nucleic acid.
  • the neutral lipid is often helpful in maintaining a stable lipid bilayer in liposomes used to make the nucleic acid:lipid complexes, and can significantly affect transfection efficiency.
  • Liposomes may have a single lipid bilayer (unilamellar) or more than one bilayer (multilamellar).
  • liposomes are typically referred to as large unilamellar vesicles (LUVs), multilamellar vesicles (MLVs) or small unilamellar vesicles (SUVs).
  • LUVs large unilamellar vesicles
  • MLVs multilamellar vesicles
  • SUVs small unilamellar vesicles
  • Cationic liposomes are typically mixed with polyanionic compounds (including nucleic acids) for delivery to cells. Complexes form by charge interactions between the cationic lipid components and the negative charges of the polyanionic compounds.
  • liposomal formulations are known and commercially available and can be tested in the assays of the present invention for precipitation, DNA protection, pH effects and the like. Because liposomal formulations are widely available, no attempt will be made here to describe the synthesis of liposomes in general. Two references which describe a number of therapeutic formulations and methods are WO 96/40962 and WO 96/40963.
  • the lipid carriers of the invention will generally be a mixture of cationic lipid and neutral helper lipid in a molar ratio of from about 3:1 to 1 :3, preferably about 1 : 1.
  • the lipid carriers may include cholesterol, DOPE (dioleoylphosphatidylethanalamine), DLPE (l,2-dilauroyl-sn-glycero-3-phosphoethanolamine), DOTMA (N-(2,3-dioleyloxy)propyl- N,N-N-triethylammonium chloride), or DiPPE (Diphytenoyl phosphatidyl ethanolamine) alone or in combination as the helper lipid, or may include additional non-cationic helper lipids, which may be either anionic or neutral lipids.
  • DOPE dioleoylphosphatidylethanalamine
  • DLPE l,2-dilauroyl-sn-glycero-3-phosphoethanolamine
  • DOTMA N-(2,
  • the lipid carriers will have, as the lipid components, a single cationic lipid and a single neutral lipid, preferably in approximately equimolar amounts.
  • Lipid mixtures typically are prepared in chloroform, dried, and rehydrated in, e.g., 5% dextrose in water or a physiologic buffer to form liposomes. Low ionic strength solutions are preferred.
  • Liposomes may be LUVs, MLVs, or SUVs.
  • the liposomes formed upon rehydration are predominantly MLVs, and SUV's are formed from them by sonication or by extrusion through membranes with pore sizes ranging from 50 to 600nm to reduce their size. Most preferably, the liposomes are extruded through a series of membranes with decreasing pore sizes, e.g., 400nm, 200nm and 50nm.
  • the resulting liposomes are mixed with a nucleic acid solution with constant agitation to form the cationic lipid-nucleic acid transfection complexes.
  • Cationic lipid-nucleic acid transfection complexes can be prepared in various formulations depending on the target cells to be transfected. While a range of lipid-nucleic acid complex formulations will be effective in cell transfection, optimal conditions are determined empirically in the desired system. Lipid carrier compositions are evaluated, e.g., by " their ability to deliver a reporter gene (e.g., CAT, which encodes chloramphenicol acetyltransferase, luciferase, ⁇ -galactosidase, or GFP) in vitro, or in vivo to a given tissue type in an animal, or in assays which test stability, protection of nucleic acids, and the like.
  • a reporter gene e.g., CAT, which encodes chloramphenicol acetyltransferase, luciferase, ⁇ -galactosidase, or GFP
  • the lipid mixtures are complexed with nucleic acids in different ratios depending on the target cell type, generally ranging from about 6:1 to 1 :20 ⁇ g nucleic acid:nmole cationic lipid.
  • Non-lipid material (such as biological molecules being delivered to an animal cell) can be conjugated to the lipid carriers through a linking group to one or more hydrophobic groups, e.g., using alkyl chains containing from about 12 to 20 carbon atoms, either prior or subsequent to vesicle formation.
  • Various linking groups can be used for joining the lipid chains to the compound. Functionalities of particular interest include thioethers, disulfides, carboxamides, alkylamines, ethers, and the like, used individually or in combination.
  • the particular manner of linking the compound to a lipid group is not a critical part of this invention, and the literature provides a great variety of such methods.
  • Formulations suitable for administration include aqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non- aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • t he formu'ations of the present invention can be administered at a rate determined by the LD-50 (or other measure of toxicity) of the cell type, and the side-effects at va ⁇ ous concentrations, as applied to the mass and overall health of the mammal Administration can be accomplished via single or divided doses
  • the vectors of this invention can supplement other treatments for a condition by known conventional therapy, including medical or surgical approaches
  • the vectors of the invention are delivered to the site of exposed subchondral cells in an artificial mat ⁇ x or plug to facilitate transfection efficiency Means for prepa ⁇ ng and de ve ⁇ ng the artificial mat ⁇ x are desc ⁇ bed below
  • any of a va ⁇ ety of genes or gene products can be used m the present invention
  • any marker gene, growth factor, cytokine antagonist or proteinase inhibitor can be used
  • any polypeptide that marks cells in vivo, or that stimulates chondrocyte or chondroprogenitor cell growth, proliferation, chondrogenic differentiation, mat ⁇ x synthesis, or any other aspect of chondrogenesis can be used
  • any of a va ⁇ ety of mtracellular proteins, as well as RNA species can be used to regulate gene expression therapeutically
  • marker gene refers to a gene whose expression allows the detection of expressing cells through any of a va ⁇ ety of means, e g visual detection
  • marker genes allow the visualization of cells for momto ⁇ ng the growth, proliferation, or survival of subchondral cells in vivo
  • Such markers could be used, e g , to monitor the efficacy of a clinical treatment, or to monitor the progress of a disease over time
  • Suitable such marker genes include ⁇ -galactosidase, alkaline phosphatase, alcohol dehydrogenase, GFP (Ikawa et al , (1999) Curr Top Dev Biol 44 1-20, Haseloff (1999) Methods Cell Biol 58 139-51, Tsien (1998) Annu Rev Biochem 67 509-44), firefly luciferase (Naylor (1999) Biochem Pharmacol 58(5) 749-57), and others
  • nucleic acids encoding growth factors are used, where "growth factor” refers to any polypeptide that modulates the rate and/or extent of cellular, tissue, or organismal growth or repair Growth factors can thus be used to facilitate the repair or regeneration of cartilage by stimulating growth and/or proliferation in subchondral cells, mat ⁇ x production, and/or the chondrocytic differentiation of chondroprogenitor or mesenchymal stem cells, either implanted or locally available in the joint. Also preferred is the introduction of genes encoding inhibitors or antagonists of cytokines and growth factors that are involved in the degeneration of cartilage. Equally useful are inhibitors of proteases that directly degrade matrix molecules or indirectly act by degrading growth factors, which would otherwise aid the repair or regeneration processes. In numerous embodiments, multiple such genes are used.
  • any of a variety of growth factors can be used to increase matrix deposition in vivo (Flechtenmacher, J. et al, Arthritis Rheum 39: 1896 (1996); Glansbeek, H.L. et al, Arthritis Rheum 40:1020 (1997); Morales, T.I. et al, J Biol Chem 263:12828 (1988); Tyler, J.A. Biochem J 260:543 (1989)).
  • TGF- ⁇ transforming growth factor- ⁇
  • TGF- ⁇ transforming growth factor- ⁇
  • TGF- ⁇ l TGF- ⁇ l
  • TGF- ⁇ 2 TGF- ⁇ 3
  • a homolog, derivative, or fragment thereof is used (see, e.g., Lafeber, FPJG et al, Br J Rheum 32:281 (1993)).
  • IGF-1 TGF-alpha
  • at least one fibroblast growth factor the bone morphogenetic proteins (BMPs) 2-12
  • BMPs bone morphogenetic proteins
  • cartilage-derived growth and differentiation factors or cartilage-derived morphogenetic protein see, e.g., Reddi, Microsc Res Tech 1998 Oct 15;43(2): 131-6; Chang et al, J Biol Chem 1994 Nov 11;269(45):28227-34; Tsumaki et al, (1999) , J Cell Biol 1999 Jan 11;144(1): 161-73
  • members of the hedgehog family see, e.g., Bianco et al, Matrix Biol 1998 Jul;17(3):185-92; Ingham, EMBO J 1998 Jul 1;17(13):3505-11
  • interleukin-1 activates chondrocytes and induces cartilage breakdown in vivo. Additionally, interleukin-1 inhibits cartilaginous matrix synthesis by chondrocytes, thereby suppressing repair of cartilage. Interleukin-1 also induces bone resorption and thus may account for the loss of bone density seen in rheumatoid arthritis.
  • polynucleotides will be used that encode proteins that inhibit one or more effects of interleukin-1 on joints, such as cartilage breakdown (see, e.g., Caron, J.P et al, Arthritis Rheum 39:1535 (1996), and U.S. Patent No. 5,858,355).
  • the interleukin-1 receptor antagonist IL-lRa, or IRAP
  • This gene is capable of binding to and neutralizing interleukin-1 in vivo. Soluble forms of IL-1R can also be used.
  • cytokines or cytokine antagonists can also be used to inhibit the catabolic activities of chondrocytes, including IL-13 (Jovanovic, D. et al, Osteoarthritis Cart6:40 (1997)), IL-4 (Yeh, L.A. et al, J Rheumatol 22: 1740 ( 1995)). and soluble forms of IL- 1 and TNT receptors.
  • Biological inhibitors of the proteinases produced by ost ⁇ oarthritic chondrocytes include the tissue inhibitors of metalloproteinases (TIMPs), plasminogen activator inhibitors, serpins and ⁇ 2 - macroglobulin, among others (Cawston, T.
  • IL-10 e.g., vIL-10 (see, e.g., Lechman et al, (1999) J Immunol 163(4):2202-8).
  • inhibitors of NO synthase, and Ik- ⁇ see, e.g., Wulczyn et al., J Mol Med 1996 Dec;74(12):749-69).
  • the present methods will be used to inhibit the expression of one or more genes.
  • Such inhibition can be accomplished, e.g., using antisense or ribozyme technology (see, e.g., Couture, L.A. et al, Trends Genet 12:510 (1996)).
  • Antisense or ribozyme nucleic acids can be administered directly or by delivery of genes encoding antisense or ribozyme molecules.
  • nucleic acids will be inserted into vectors using standard molecular biological techniques.
  • Vectors may be used at multiple stages of the practice of the invention, including for subcloning nucleic acids encoding, e.g., components of proteins or additional elements controlling protein expression, vector selectability, etc.
  • Vectors may also be used to maintain or amplify the nucleic acids, for example by inserting the vector into prokaryotic or eukaryotic cells and growing the cells in culture.
  • introduction of the present nucleic acids into cells in vivo will be achieved using viral vectors.
  • viral vectors derived from retroviruses, adenovirus, adeno-associated virus, or herpes simplex virus have found use in human clinical trials, and can be used herein.
  • Retroviruses and adeno-associated viruses offer particular advantages in the pursuit of long term gene expression because they integrate their genetic material into the chromosomal DNA of the cells they infect.
  • the viruses are genetically disabled so that they cannot replicate or cause disease, yet can still transfer genes effectively.
  • a type of retrovirus known as a lentivirus is able to transduce non-dividing cells, and can also be used.
  • Vectors derived from adenoviruses are increasingly popular, because the viral vectors can be grown to high titer, are able to contain large segments of DNA, and are highly infectious to both dividing and non-dividing cells. The latter property facilitates their use during in vivo protocols where the gene is delivered directly to the patient. See, Rosenfeld, M. A., et al, 1992, Cell, 68:143155; Jaffe, H. A. et al, 1992; Nature Genetics 1.372-378; Lemarchand, P. et al, 1992, Proc. Natl. Acad. Sci. USA, 89:6482-6486.
  • Adeno-associated virus can also be used, as it causes no known diseases and integrates at a precise location into the chromosomal DNA of the cells it infects. See, e.g., Xiao et al, J Virol 1999 May;73(5):3994-4003; Prince, Pathology 1998 Nov;30(4):335- 47; Rabinowitz and Samulski, Curr Opin Biotechnol 1998 Oct;9(5):470-5).
  • viral-based and nonviral, e.g., plasmid-based, expression systems are provided.
  • Nonviral vectors and systems include plasmids and episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al., 1997, Nat Genet 15:345).
  • plasmids useful for expression of polynucleotides and polypeptides in mammalian (e.g., human) cells include pcDNA3.1/His, pEBVHis A, B & C, (Invitrogen, San Diego CA), MPSV vectors, others described in the Invitrogen 1997 Catalog (Invitrogen Inc, San Diego CA), which is incorporated herein in its entirety by reference, and numerous others known in the art.
  • Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Ban virus, vaccinia virus vectors and Semliki Forest virus (SFV).
  • SFV and vaccinia vectors are discussed generally in Ausubel et al, supra, Ch. 16. These vectors are often made up of two components, a modified viral genome and a coat structure surrounding it (see generally, Smith, 1995, Ann. Rev. Microbiol 49: 807), although sometimes viral vectors are introduced in naked form or coated with proteins other than viral proteins.
  • the viral nucleic acid in a vector may be changed in many ways, for example, when designed for gene therapy.
  • the goals of these changes are to disable growth of the virus in target cells while maintaining its ability to grow in vector form in available packaging or helper cells, to provide space within the viral genome for insertion of exogenous DNA sequences, and to incorporate new sequences that encode and enable appropriate expression of the gene of interest.
  • Viral vector nucleic acids generally comprise two components: essential cis - acting viral sequences for replication and packaging in a helper line and the transcription unit for the exogenous gene. Other viral functions are expressed in trans in a specific packaging or helper cell line.
  • Adenoviral vectors e.g., for use in human gene therapy
  • a sequence may be ligated into an adenovirus transcription translation complex consisting of the late promoter and tripartite leader sequence.
  • Insertion in a nonessential El or E3 region of the viral genome will result in a viable virus capable of expressing in infected host cells (Logan and Shenk, 1984, Proc. Natl. Acad. Sci., 81 :3655).
  • Replication-defective retroviral vectors harboring a therapeutic polynucleotide sequence as part of the retroviral genome are described in, e.g., Miller et al, 1990, Mol. Cell. Biol. 10: 4239; Kolberg, 1992, J. NIH Res. 4: 43; and Cometta et al, 1991, Hum. Gene Ther. 2: 215.
  • the surface of the virus can be coated, e.g., by covalent attachment, with polyethylene glycol (PEG; see, e.g., O'Riordan et al, (1999) Hum Gene Ther. 10(8): 1349-58.).
  • PEG polyethylene glycol
  • Such "PEGylation" of viruses can impart various benefits, including increasing the infectivity of the virus, and lowering the host immune response to the virus.
  • promoters capable of driving gene expression in vivo or in vitro can be used, in mammalian cell systems promoters from mammalian genes or from mammalian viruses are often appropriate. Suitable promoters may be constitutive, cell type-specific, stage-specific, and or modulable or regulable (e.g., by hormones such as glucocorticoids).
  • Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline - inducible CMV promoter (such as the human immediate - early CMV promoter), the constitutive CMV promoter, and promoter - enhancer combinations known ' in the art.
  • the vectors of this invention can be administered in any format, the vectors are typically formulated in an artificial matrix to enhance transfection efficiency.
  • the matrices used to deliver the polynucleotide formulations according to the present methods are comprised of natural components, e.g., collagen, they are referred to herein as "artificial matrices" to distinguish them from endogenous matrix, e.g., collagen matrix, present in natural cartilage.
  • the artificial matrix is easy to handle, porous, spongy, malleable, and resorbable by the body.
  • suitable artificial matrix materials are known to those of skill in the art.
  • type I collagen can be used as the artificial matrix material.
  • artificial matrices can be made, e.g., from type II collagen or other types of collagen, hyaluronan or other glycosaminoglycans, fibrin, various synthetic biodegradable polymers, and mixtures of the above. See, e.g, Riesle et al, J Cell Biochem 1998 Dec 1 ;71(3):313-27.
  • the vector solution is mixed with a collagen solution and allowed to gel.
  • collagen can be dipped in a solution of plasmid DNA and inserted into the perforated bone (see, e.g., Fang et al, Proc Natl Acad Sci USA 93(12):5753-8); Bonadio and Fang, WO 98/22492).
  • Pharmaceutical Compositions e.g., Fang et al, Proc Natl Acad Sci USA 93(12):5753-8; Bonadio and Fang, WO 98/22492.
  • nucleic acid vectors of this invention can be formulated as pharmaceutical compositions for local administration to joints according to well-known techniques. Actual methods for preparing appropriate compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania (1980).
  • compositions are administered to a patient suffering from a disease or injury in an amount sufficient to cure or at least partially a ⁇ est the disease or injury and its complications.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease- and the general state of the patient's health. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the molecules described herein to effectively treat the patient.
  • the toxicity and therapeutic efficacy of the nucleic acids provided by the invention are determined using standard pharmaceutical procedures in cell cultures or experimental animals.
  • the therapeutic index (LD 5 o/ED 50 ) can be determined from these experiments.
  • a single dose of nucleic acids will be applied to the exposed subchondral cells, although in certain cases multiple doses will be applied.
  • a typical pharmaceutical composition of the invention would depend on the particular gene, patient, medical condition, pharmaceutical composition, and other factors, but will typically be from about 10 ng to about 10 mg, preferably between about 10 ⁇ g and about 1 mg of nucleic acid, e.g., plasmid or vector DNA.
  • nucleic acid e.g., plasmid or vector DNA.
  • plasmid or vector DNA e.g., plasmid or vector DNA.
  • the vectors of the invention can be administered either alone or in conjunction with other well-known therapies.
  • the present methods can be used to express any of a number of chondrogenesis- promoting proteins in subchondral cells exposed by a perforation in a subchondral bone in a joint.
  • chondrogenesis-promoting proteins can be used to promote the growth and/or proliferation of the cells (e.g., using growth factors), the chondrogenic differentiation of the cells (e.g-., using morphogenetic or other factors), matrix production by the cells (e.g., using morphogenetic, growth, and/or other factors), and can be used to inhibit cartilage degradation in the joint (e.g., using cytokine and/or proteinase inhibitors).
  • the present methods can be used to promote chondrogenesis in a joint, where "chondrogenesis” refers to the ability to promote cartilage growth in a joint, e.g., by promoting chondrocyte growth, proliferation, and/or differentiation, by promoting matrix synthesis, or inhibiting matrix degradation in a joint.
  • chondrogenesis refers to the ability to promote cartilage growth in a joint, e.g., by promoting chondrocyte growth, proliferation, and/or differentiation, by promoting matrix synthesis, or inhibiting matrix degradation in a joint.
  • the present methods can be used to promote chondrogenesis within a damaged joint, and can thus be used to treat any of a large number of conditions.
  • any condition for which a net increase in cartilage production would be beneficial can be treated.
  • any inflammatory or degenerative condition causing cartilage loss or damage can be treated, such as any type of arthritis, including osteoarthritis, rheumatoid arthritis, gout, and others.
  • Other conditions include osteochondritis dissecans, avascular necrosis, chondromalacia, and others. See, e.g., information provided by the Arthritis Foundation (www.arthritis.org) or the Wheeless' Textbook of Orthopaedics (http://www.medmedia.com/med.htm).
  • the present methods would allow the regeneration of both cartilage and subchondral bone.
  • cartilage defects resulting from an injury to the cartilage e.g., from major trauma, minor trauma, repeated minor trauma, surgery, e.g., producing rotational force, shear force, etc., and producing any type of lesion or damage, such as fibrillation, discoloration, softness, cracks, tears, craters, etc.
  • Both full-thickness and partial-thickness defects are treatable using the present methods.
  • the invention also provides packs, dispenser devices, and kits for administering vectors of the invention to a mammal.
  • packs or dispenser devices that contain one or more unit dosage forms are provided.
  • instructions for administration of the nucleic acids will be provided with the packaging, along with a suitable indication on the label that the nucleic acid is suitable for treatment of an indicated condition.
  • the label may state that the active compound within the packaging is useful for treating osteoarthritis, or for preventing or treating other diseases or conditions that are associated with dest ⁇ iction of connective tissue.
  • Figure 2 shows the results of implantation of numerous non-viral formulations mixed with a Type I and Type 2 collagen matrix.
  • the formulations shown in Figure 2 include: naked DNA (2 mg/ml plasmid DNA); MB 192 - 1 :1 (DNAxationic lipid) DOTIM CHOLESTEROL Liposomes (2 mg/ml final DNA concentration); MB322 -1 :1 (DNA hitosan) Glycol chitosan - 1 mg/ml final DNA concentration; MB314 — 3:1 (DNA:paromomycin) Paromomycin - 1 mg/ml final DNA concentration; MB306 — 1 :40:0.1 :0.6 (DNA:Tributyrin:RGDS peptide:Brij 35 - 2 mg/ml DNA final concentration; and a control (Phosphate buffered saline (PBS)).
  • PBS Phosphate buffered saline
  • IGF-1 insulinlike growth factor 1
  • TGF ⁇ l transforming growth factor beta 1
  • ⁇ E1 ⁇ E3, serotype 5 adenoviral vectors were used.
  • Transgenes encoding human IGF-1, TGF ⁇ l, or, as a control, the lac Z gene of Escherichia coli, were inserted into the El region of the virus by cre-lox recombination (Hardy, et al. (1997) 71 :1842-9). In each case, gene expression was driven by the human cytomegalovirus early promoter.
  • the resulting vectors were designated AdIGF-1, AdTGF ⁇ l, and AdLacZ, respectively.
  • Articular chondrocytes were isolated from articular cartilage recovered from the knee and shoulder joints of skeletally mature NZW rabbits, using described methods (Green WT (1971) Clin Orthop 75:248-60). Cells were seeded at a density of 2 x 10 4 cells/well into monolayer cultures on 24-well plates using Ham's F-12 nutrient medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified atmosphere of 5% CO at 37°C. The cultures became confluent after 7-10 days, and typically contained 2 x 10 5 cells/well.
  • FBS fetal bovine serum
  • penicillin-streptomycin penicillin-streptomycin
  • GBSS sterile Gey's balanced salt solution
  • adenoviruses that contained genes encoding IGF-1, TGFl ⁇ , or ⁇ - galactosidase (lac Z g ⁇ ne).
  • Transduction was performed in 300 microliters of GBSS for 1 hour at 37°C at various multiplicities of infection (MOIs), as indicated.
  • MOIs multiplicities of infection
  • the supernatant was aspirated and replaced with 500 microliters of Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% F3S and antibiotics.
  • DMEM Dulbecco's modified Eagle's medium
  • Exogenous recombinant human IGF-1 (R&D Systems. Minneapolis, MN) was added to the media of the nontransduced control groups.
  • Recombinant human IL-l- ⁇ (2 ng/ml) was also added to the appropriate groups at this time. Cells were then incubated for 48 hours before the addition of radionucleotides for the determination of matrix synthesis.
  • the concentration of IGF-1 in cell supematants was determined at 48 hours following transduction using a commercially available enzyme-linked immunosorbent assay (ELISA) kit and protocol (Diagnostics Laboratory Systems, Webster, TX). Proteoglycan and collagen synthesis.
  • ELISA enzyme-linked immunosorbent assay
  • proteoglycan synthesis To measure proteoglycan synthesis, the media were aspirated following the initial 48-hour incubation period and replaced with 500 microliters of fresh DMEM with 1% FBS. Exogenous growth factors and IL-1 were also readministered to the appropriate wells. Each culture well received 20 microCi/ml of 5 STabeled Na 2 SO , and the cells were incubated for an additional 24 hours. The synthesis of proteoglycans was determined in the cell layer and the media by the incorporation of 35 SO 4 2" into glycosaminoglycans. Size-exclusion chromatography with PD10 columns (Pharmacia,
  • Protein synthesis was determined by the incorporation of H-proline into collagen and noncollagenous proteins in the following manner.
  • the medium was removed and replaced with serumless medium containing 50 micrograms/ml of ascorbic acid, 50 micrograms/ml of ⁇ -aminopropionitrile, and 20 microCi/ml of 3 H-proline for 24 hours.
  • serumless medium containing 50 micrograms/ml of ascorbic acid, 50 micrograms/ml of ⁇ -aminopropionitrile, and 20 microCi/ml of 3 H-proline for 24 hours.
  • the conditioned medium and cell layer were collected and combined, and the relative collagen synthesis was determined by 3 H-proline incorporation using a modified collagenase digestion method.
  • a carrier protein (2 mg ml of pepsin-solubiliz ⁇ d bovine type II collagen) was added, and the proteins were precipitated by addition of 10% trichloroacetic acid (TCA).
  • TCA trichloroacetic acid
  • the TCA- precipitated proteins were recovered by centrifugation at 14,000 revolutions per minute for 30 minutes at 4°C.
  • the recovered protein pellets were washed repeatedly with 5% TCA to remove the unbound isotope.
  • the washed pellets were resuspended in the collagenase buffer (5 millimolar CaCl? and 3 millimolar N-ethylmaleimide), the pH was adjusted to 7, and the suspensions were incubated at 37°C for 2 hours.
  • TCA was then added, and the mixture was c ⁇ ntrifuged for 30 minutes at 14,000 rpm.
  • the supematants were removed and were used as enzyme blanks.
  • the pellets were resuspended in the collagenase buffer, the pH was adjusted to 7 by the addition of NaOH, and purified bacterial collagenase ABC form III (Advanced Biofactures, Lynbrook, NY) was added (20 units/ml) and incubated for 2 hours at 37°C.
  • the collagenase-digestible proteins were separated from nondigested proteins by the addition of 5% TCA followed by centrifugation at 14,000 rpm for 30 minutes.
  • the pellets were redissolved in 0.2 M NaOH, and aliquots of the pellets and the radioactivities of the supematants were measured to determine the level of collagen and noncollagenous protein synthesis.
  • the data were co ⁇ ected for cell number by counting cells in parallel wells that received the same factors and were plated at the same density as in the experimental wells.
  • Collagen phenotyping was also performed on each sample, as described elsewhere (Cao, et al. (1997) Biochem J 325:305-10.) Briefly, following the 24-hour labeling period with 3 H-proline, all samples underwent pepsin (100 micrograms/ml) digestion for 4 hours. The pepsin-resistant chains were precipitated with 3 M NaCl followed by dialysis against NH HCO for 5 days. Samples were then dried and separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels were treated with EN 3 HANCE (DuPont NEN, Boston, MA) prior to autoradiography.
  • EN 3 HANCE DuPont NEN, Boston, MA
  • IGF-1 gene expression and matrix synthesis IGF-1 gene expression and matrix synthesis.
  • Figure 3 shows the production of insulin-like growth factor 1 (IGF-1) by transduced chondrocytes and its effects on matrix synthesis.
  • Figure 3 A shows IGF-1 concentrations in conditioned media following transduction with the IGF-1 gene or the addition of IGF-1 protein. Cultures of chondrocytes were infected with AdIGF-1 at the indicated multiplicity of infection (MOI) or were supplemented with IGF-1 at the indicated dosages. After 48 hours, all conditioned media were assayed for IGF-1.
  • Figure 3B shows effects of IGF-1 protein and gene on the synthesis of proteoglycans by chondrocytes.
  • IGF-1 protein had a more modest effect on the synthesis of collagen and noncollagenous proteins by the chondrocytes, but the stimulatory effects of transducing the IGF-1 cDNA were very marked (Figure 3C). Even at an MOI of 100, where IGF-1 accumulated in the medium to ⁇ 80 ng ml ( Figure 3A), an approximately 3-fold increment was seen ( Figure 3C). -Addition of 100 ng/ml of protein, in contrast, achieved only about a 1.5-fold increment, despite the presence of -160 ng/ml IGF-1 in the culture medium ( Figure 3 A).

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Abstract

La présente invention concerne des procédés destinés à des méthodes de thérapie génique in vivo de traitement de maladies articulaires telles que l'ostéroarthrite. Les procédés consistent en l'exposition de cellules sous-chondrales dans une articulation et ensuite la transfection de ces cellules in vivo.
PCT/US2000/022801 1999-08-20 2000-08-18 Procedes d'administration de genes in vivo a des sites de deterioration de cartilage WO2001013960A1 (fr)

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AU69193/00A AU6919300A (en) 1999-08-20 2000-08-18 Methods for in vivo gene delivery to sites of cartilage damage

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US15011899P 1999-08-20 1999-08-20
US60/150,118 1999-08-20

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001082973A3 (fr) * 2000-04-28 2003-02-06 Univ Pittsburgh Vecteurs viraux et non viraux en tant que vehicules d'administration de transgenes pour le traitement de pathologies osseuses
WO2002083080A3 (fr) * 2001-04-17 2003-02-20 Genetix Pharmaceuticals Inc Methode de traitement de l'arthrite utilisant des vecteurs lentiviraux en therapie genique
EP1530972A2 (fr) 2003-11-17 2005-05-18 University of Iowa Research Foundation Inc. L'utilisation d'un agent préparé d'un parasite pour la prévention et le contrôle de maladies
EP1485487A4 (fr) * 2002-03-12 2005-11-09 Tissuegene Inc Regeneration de cartilages a l'aide de chondrocytes et de tgf-beta
US20110129534A1 (en) * 2003-11-24 2011-06-02 Canji, Inc. Reduction of dermal scarring
WO2015035395A1 (fr) * 2013-09-09 2015-03-12 Figene, Llc Thérapie génique pour la régénération de cellules de type chondrocytes ou cartilagineuses

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WO1992011359A1 (fr) * 1990-12-20 1992-07-09 University Of Pittsburgh Of The Commonwealth System Of Higher Education Gene recepteur tronque d'interleukine-1 utilise pour le traitement de l'arthrite
US5766585A (en) * 1993-12-14 1998-06-16 University Of Pittsburgh Of The Commonwealth System Of Higher Education Systemic gene treatment of connective tissue diseases with IRAP-1
US5770580A (en) * 1992-04-13 1998-06-23 Baylor College Of Medicine Somatic gene therapy to cells associated with fluid spaces
US6077987A (en) * 1997-09-04 2000-06-20 North Shore-Long Island Jewish Research Institute Genetic engineering of cells to enhance healing and tissue regeneration

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WO1992011359A1 (fr) * 1990-12-20 1992-07-09 University Of Pittsburgh Of The Commonwealth System Of Higher Education Gene recepteur tronque d'interleukine-1 utilise pour le traitement de l'arthrite
US5770580A (en) * 1992-04-13 1998-06-23 Baylor College Of Medicine Somatic gene therapy to cells associated with fluid spaces
US5766585A (en) * 1993-12-14 1998-06-16 University Of Pittsburgh Of The Commonwealth System Of Higher Education Systemic gene treatment of connective tissue diseases with IRAP-1
US6077987A (en) * 1997-09-04 2000-06-20 North Shore-Long Island Jewish Research Institute Genetic engineering of cells to enhance healing and tissue regeneration

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Title
GHIVIZZANI ET AL.: "Direct adenovirus-mediated gene transfer of interleukin 1 and tumor necrosis factor alpha soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects", PROC. NATL. ACAD. SCI. USA, vol. 95, April 1998 (1998-04-01), pages 4613 - 4618, XP002933069 *
SONG ET AL.: "Plasmid DNA encoding transforming growth factor-B1 supresses chronic disease in a streptococcal cell wall-induced arthritis model", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 101, no. 12, June 1998 (1998-06-01), pages 2615 - 2621, XP002933068 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001082973A3 (fr) * 2000-04-28 2003-02-06 Univ Pittsburgh Vecteurs viraux et non viraux en tant que vehicules d'administration de transgenes pour le traitement de pathologies osseuses
WO2002083080A3 (fr) * 2001-04-17 2003-02-20 Genetix Pharmaceuticals Inc Methode de traitement de l'arthrite utilisant des vecteurs lentiviraux en therapie genique
EP1485487A4 (fr) * 2002-03-12 2005-11-09 Tissuegene Inc Regeneration de cartilages a l'aide de chondrocytes et de tgf-beta
EP1530972A2 (fr) 2003-11-17 2005-05-18 University of Iowa Research Foundation Inc. L'utilisation d'un agent préparé d'un parasite pour la prévention et le contrôle de maladies
EP1530972A3 (fr) * 2003-11-17 2007-06-13 University of Iowa Research Foundation Inc. L'utilisation d'un agent préparé d'un parasite pour la prévention et le contrôle de maladies
US20110129534A1 (en) * 2003-11-24 2011-06-02 Canji, Inc. Reduction of dermal scarring
US8329671B2 (en) * 2003-11-24 2012-12-11 Canji, Inc. Reduction of dermal scarring
WO2015035395A1 (fr) * 2013-09-09 2015-03-12 Figene, Llc Thérapie génique pour la régénération de cellules de type chondrocytes ou cartilagineuses
CN105636614A (zh) * 2013-09-09 2016-06-01 菲格内有限责任公司 用于软骨细胞或软骨型细胞再生的基因治疗
US20160220699A1 (en) * 2013-09-09 2016-08-04 Figene, Llc Gene therapy for the regeneration of chondrocytes or cartilage type cells
AU2014317861B2 (en) * 2013-09-09 2019-11-28 Figene, Llc Gene therapy for the regeneration of chondrocytes or cartilage type cells
US11819555B2 (en) 2013-09-09 2023-11-21 Figene, Llc Gene therapy for the regeneration of chondrocytes or cartilage type cells

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