WO2000034500A2

WO2000034500A2 - Methods for increasing the efficiency of gene transfer

Info

Publication number: WO2000034500A2
Application number: PCT/US1999/028640
Authority: WO
Inventors: Francois Binette; Maria E. Brown
Original assignee: Genzyme Corporation
Priority date: 1998-12-04
Filing date: 1999-12-03
Publication date: 2000-06-15
Also published as: AU2038900A; WO2000034500A3

Abstract

The invention relates to compositions and methods for increasing the efficiency of gene transfer into cells surrounded by an extracellular matrix-like material. The compositions and methods comprise an agent that degrades the extracellular matrix-like material. The agent may be administered prior to or at the time of gene transfer.

Description

METHODS FOR INCREASING THE EFFICIENCY OF GENE TRANSFER

Technical Field of the Invention This invention relates to methods for increasing the efficiency of gene transfer into cells embedded within an extracellular matrix-like material.

Background of the Invention

Gene transfer vehicles such as viral vectors, plasmids (i.e., naked DNA), liposomes, DNA-protein conjugates, and others, have been used succesfully to deliver heterologous nucleic acids to a wide variety of cells in vitro and in vivo. Certain cell types, however, are resistant to introduction of transgene-carrying vectors, regardless of the route of delivery or delivery vehicle. In other cells, while gene transfer can be accomplished at low levels, the efficiency of gene transfer is not high enough to allow the transferred gene to produce the desired effect.

One example of cells refractory to gene transfer are chondrocytes — the cells which make up cartilage tissue. Articular cartilage, found at the end of articulating bones, is a specialized tissue responsible for elasticity, resistance to compressive forces, and the smooth gliding that is characteristic of healthy joint function. Cartilage is composed of chondrocytes embedded in a hydrated extracellular matrix rich in collagens (predominantly type II) and proteoglycans (predominantly aggrecan). The matrix macromolecules are synthesized by the chondrocytes.

Methods which would increase the efficiency of gene transfer into chondrocytes and other cells embedded within tissues is an important aim in the fields of in vivo tissue repair, cancer, and other diseases amenable to treatment or prevention with gene therapy. In addition, the resilience of certain cells to gene transfer in vitro has hindered in vitro efforts to study the control of cellular growth and differentiation. Such efforts would be greatly forwarded by the ability to introduce heterologous genetic material into the cells while present in tissues such as cartilage explants or other in vitro conditions in which an extracellular matrix material is present.

Summary of the Invention

This invention is based, in part, on the discovery that the resistance of chondrocytes to infection with adenoviruses is due to the physical and biochemical barrier provided by the extracellular matrix in which they reside. When the barrier is lessened, the efficiency of gene transfer may be increased. These findings are applicable not only to cartilage, but also to other tissues in which the physical and biochemical environment in which the target cells reside affects the efficiency of gene transfer into such cells.

In one embodiment, the invention provides methods for increasing the efficiency of gene transfer into cells surrounded by an extracellular matrix-like material comprising the step of pretreating the cells with an agent which degrades the extracellular matrix or other physical barrier to gene delivery. Once degradation of the extracellular matrix-like material has been initiated, the cells are subjected to gene transfer.

In a preferred embodiment, a method is provided which renders chondrocytes susceptible to in vivo gene transfer using adeno viral vectors. This method comprises the step of pretreatment of the cartilage tissue with agents which degrade the proteoglycans which make up the extracellular matrix. In another embodiment, the efficiency of gene transfer into chondrocytes using non- adenoviral vectors is increased by treating the cells with an agent which degrades the extracellular matrix.

In another embodiment, this invention relates to compositions which are useful to increase the efficiency of gene transfer, which compositions comprise agents which are effective to cause degradation of the extracellular matrix-like material in which the target cells reside.

In another embodiment, this invention relates to cells and tissues which have been subjected to gene transfer after treatment according to the methods of this invention.

Detailed Description of the Invention

This invention is directed to methods and compositions for increasing the efficiency of gene transfer into cells which are present in an environment that presents a physical or biochemical barrier to gene transfer. Specifically, we have discovered that cells which are associated with an extracellular matrix are refractory to gene transfer using viral vectors due to the integrity of the matrix in which they reside. According to the methods of this invention, when such matrix is degraded or dissociated, for example with enzymes which degrade proteoglycans, efficient gene transfer may be obtained. Without being bound by theory, we believe that the extracellular matrix may serve to hinder gene transfer by providing a biochemical and physical barrier to passive diffusion of the gene transfer vehicle. While these findings were initially made with cartilage tissue, they are applicable to a wide variety of cells and tissues in which the efficiency of gene transfer, regardless of the gene transfer vehicle employed, may be increased by subjecting the cells to agents which lessen the physical and/or biochemical barriers to gene transfer. Such agents are those which degrade the extracellular matrix-like material in which the target cells reside.

According to the methods of this invention, the efficiency of gene transfer into cells surrounded by an extracellular matrix-like material is increased by treating the cells with an agent that degrades the extracellular matrix-like material prior to gene transfer. Such methods are useful to increase the efficiency of gene transfer in vitro and in vivo.

Thus, in one embodiment, a method is provided whereby the efficiency of gene transfer into cells present in an extracellular matrix-like material is increased, the method comprising the steps of : (a) treating the cells with an effective amount of an agent that degrades the extracellular matrix-like material under conditions sufficient to cause degradation of the extracellular matrix-like material; and (b) subjecting the cells to gene transfer.

The compositions and methods of this invention are useful to increase the efficiency of gene transfer both in vivo and in vitro. Thus, they are useful when gene transfer is employed in laboratory research as well as in ex vivo and in vivo gene therapy techniques.

In another embodiment, this invention provides cells in which gene transfer has been accomplished subsequent to treatment with an agent that degrades the extracellular matrix-like material in which they reside. Such cells may be, for example, chondrocytes present in cartilage tissue grown in vitro which will be implanted into a cartilage defect in a subject. In another embodiment, this invention provides methods for in vivo gene therapy directed to target cells embedded within an extracellular matrix or other physical barrier to entry of a transgene-carrying vector. Such methods comprise the step of administering to the target tissue, prior to introduction of the gene therapy vector, an effective amount of an agent which degrades the extracellular matrix-like material in which the target cells are embedded.

In another embodiment, this invention provides methods for ex vivo gene therapy directed to treatment of target cells rendered resilient to introduction of transgene-carrying vectors due to the state in which the cells have been isolated or maintained.

In another embodiment, this invention provides methods useful to increase the efficiency of gene transfer in vitro. Such methods are useful across a broad spectrum of disciplines which employ techniques involving the introduction of heterologous nucleic acids into cells present in an environment which would otherwise hinder efficient gene transfer.

In another embodiment, this invention provides compositions useful for increasing the efficiency of gene transfer, which compositions comprise an effective amount of an agent capable of degrading extracellular matrix-like material, either with or without a gene transfer agent.

As used herein, "extracellular matrix-like material" refers to any tissue extracellular matrix material which comprises proteins and proteoglycans and which provides a physical barrier to entry of gene delivery vehicles. The methods of this invention are applicable to any cell type which resides, in vitro or in vivo, in an environment in which it is surrounded by an extracellular matrix-like material.

The skilled artisan will readily recognize the types of cells and tissues amenable to the methods disclosed herein. Nevertheless, the presence of extracellular matrix-like material according to the definition presented herein may be confirmed by a variety of techniques. For example, the presence of protein and preoteoglycan is easily determined by standard biochemical and immunoassays. The characteristic of providing a physical barrier to entry of gene delivery vehicles may be determined, e.g., by plating the target cells in monolayer or other culture conditions in which the extracellular matrix-like material is not present. If the efficiency of gene transfer into the cells in the absence of the extracellular matrix-like material is higher than that achieved in the presence of such material, then the material is an extracellular matrix-like material according to the definition provided herein.

Examples of cells which are embedded in extracellular matrix-like material and which may most benefit from the methods of this invention include, but are not limited to, chondrocytes and hypertrophic chondrocytes surrounded by an extracellular matrix composed of collagen and proteoglycans, osteoblasts surrounded by collagen, proteoglycans and associated mineral deposits, and synovial cells surrounded by collagen and proteoglycan.

The methods of this invention are also useful to increase the efficiency of gene transfer into cells present in non-cartilagenous tissues which contain an extracellular matrix-like material which comprises proteins and proteoglycans and which provides a physical barrier to entry of gene delivery vehicles. Examples include, but are not limited to the dermis, in which the cells are present in an extracellular matrix of collagen and proteoglycan, as well as vascular cells and cardiac and skeletal muscle cells. Degradation of the extracellular matrix-like material may be accomplished with any agent capable of digesting or cleaving the components of the extracellular matrix-like material to the extent that the efficiency of gene transfer is enhanced. In a preferred embodiment, the proteoglycans of the extracellular matrix-like material are degraded enzymatically, e.g., with hyaluronidase, which cleaves the HA backbone of the proteoglycans. Other useful enzymatic agents include, but are not limited to aggrecanase, chondroitinase, trypsin and other proteolytic enzymes as well as combinations of the above.

As used herein, an "effective amount" of an agent that degrades the extracellular matrix-like material refers to an amount which increases the efficiency of gene transfer into target cells over the efficiency obtained by the same delivery vehicle in the absence of such treatment. Given the teachings disclosed herein, the skilled artisan may determine the effective amount of a given agent by consideration of such factors as the gene delivery vehicle, the physical and biochemical condition of the tissue to be treated, the duration of treatment, the route of administration, and the age, weight, extent of disease and physical condition of the subject being treated. Similarly, the optimum time for exposure of the target cells to the degrading agent would be expected to vary depending on the selected gene transfer agent, target cell and degrading agent, and may be determined by the ordinarily skilled artisan in accordance with the teachings provided herein.

According to the invention, the cells are treated with the extracellular matrix-like degrading agent under conditions sufficient to cause degradation of the extracellular matrixlike material, which degradation results in an increase in the efficiency of gene transfer. The skilled artisan will recognize that modifications of the methods provided herein may be desirable depending upon the specific gene transfer agent, target cell and environmental conditions in which gene transfer is desired. For example, under certain conditions, it may be desireable to expose the target cells to the degrading agent for a period of time prior to exposure to gene transfer agent, whereas under other conditions, it may be desirable to have the degrading agent present at the same time as the gene transfer agent. Such variations are within the scope of the present invention so long as the degradative process has been initiated to the extent that the efficiency of gene transfer into cells surrounded by an extracellular matrix-like material is increased.

Where exposure to the degrading agent is desired prior to introduction of the gene transfer agent, it may be desirable to remove the degrading agent either partially or completely before the gene transfer agent is introduced. Such removal may be accomplished by a variety of techniques know to the ordinarily skilled artisan, e.g., by rinsing or flushing the cells with a solution of saline or other physiologically compatible components, or by adding to the cells a component which serves to dilute the activity of the degrading agent.

Where the presence of the degrading agent is desired at the same time as the gene transfer agent, the agents may be administered in a single solution or separately. Thus, in another embodiment, this invention provides compositions for efficient gene transfer which comprise an agent which degrades an extracellular matrix-like material. In another embodiment, such compositions additionally comprise a gene transfer agent. The compositions of the present invention are useful for increasing the efficiency of gene transfer into any cells which reside in tissues or environments in which an extracellular matrix-like material inhibits the efficiency of gene transfer.

The methods of this invention are useful to increase the efficiency of gene transfer in vivo when effected by a variety of routes including intra-muscular, intravenous, intranasal, subcutaneous, intubation, lavage and intra-arterial delivery. Such methods are well known to the skilled artisan. The methods of this invention are particularly advantageous when delivery of a transgene is targetted to a particular tissue which comprises extracellular matrix-like material, e.g., cartilage.

In another embodiment, this invention provides methods for ex vivo gene therapy directed to treatment of target cells rendered resilient to introduction of transgene-carrying vectors due to the state in which the cells have been isolated or maintained. Such techniques are useful, for example, when isolated cells have been maintained or placed into a culture medium or biodegradable matrix which inhibits their penetration by transgene-carrying agents. These methods include the steps of (a) treating the isolated cells with an extracellular matrix- degrading agent, (b) introducing the transgene-carrying vector into the cells, and (c) implanting the cells into a subject.

In a related embodiment, this invention provides methods useful to increase the efficiency of gene transfer into cells in vitro. Such methods are useful across a broad spectrum of disciplines which employ techniques involving the introduction of heterologous nucleic acids into cells present in an environment which would otherwise hinder efficient gene transfer. For example, those working in the field of tissue repair may have occasion to study the behavior of certain cells while present in a variety of matrices having different structural and biochemical properties. The methods of this invention may aid such endeavors by providing a means to increase the efficiency of gene transfer in any situation in which the matrix in which the cell resides affects the efficiency of gene transfer, regardless of whether such matrix is natural or synthetic.

As used herein, the term "gene transfer" is intended to mean the introduction of a nucleic acid molecule into a target cell by any method, e.g., including by transfection, transformation, electroporation, and direction injection of DNA, and in any delivery vehicle including plasmids, viral vectors, lipid-DNA complexes, protein-DNA complexes, and any other delivery vehicle. The methods of this invention are particularly useful for gene delivery methods wherein the gene transfer vehicle is injected directly into the target tissue. The term "gene transfer agen 'similarly encompasses any agent capable of effectuating transfer of nucleic acids into a cell.

As used herein, the term "nucleic acids" encompasses RNA as well as single and double-stranded DNA, cDNA and oligonucleotides, e.g., sense and antisense oligonucleotides. When expression within the target cell is desired, the nucleic acid will be linked to expression control sequences within the delivery vehicle or vector e.g., promoters, enhancers, response elements, signal sequences, polyadenylation sequences, and the like, such that the desired expression product is produced in the target cell at a desired level and duration after gene transfer is effected according to the methods disclosed herein.

The methods of this invention may be useful to treat, prevent or study a variety of disease states involving cells refractory to gene transfer due to their physical surroundings. For example, gene transfer into chondrocytes may be useful to treat diseases affecting cartilage tissue such as rheumatoid and osteoarthritis, including familial osteoarthritis. Other diseases particularly well suited for treatment according to these methods include osteochondritis dissicans, bone edema and growth plate disorders. Another disease well suited to application of the methods disclosed herein is cystic fibrosis (CF). Specifically, the epithelial cells which are the target of gene therapy approaches to CF are surrounded by a build-up of extracellular matrix-like material consisting of mucous containing protein and proteoglycan secreted by the cells. Pretreatment of the targeted cells with an agent which would degrade the mucous, e.g., by inhalation of hyaluronidase, would be expected to increase the efficiency of gene transfer into such cells. Examples of transgenes that may be particularly useful in the methods described herein include various growth factors, differentiation factors, or chemotactic agents which aid in tissue growth or differentiation or accelerate or modulate tissue repair. Such factors include, in particular, members of the transforming growth factor β (TGFβ) family, members of the fibroblast growth factor (FGF) family, members of the insulin-like growth factor (IGF) family, CTGF and bone morphogenic proteins, growth hormone, members of the hedgehog family, tumor suppressor genes and anti-inflammatory genes such as IL-1 receptor antagonist protein (IRAP). Other useful transgenes include those capable of correcting inherited genetic deficiencies such as cystic fibrosis and Duchenne's muscular dystrophy.

The methods and compositions of this invention are of particular interest when employed to increase the efficiency of gene transfer in therapies employed specifically to inhibit the deleterious accumulation of extracellular matrix-like material. For example, introduction of TGF-β antagonists by gene therapy may be useful to treat or prevent various fibrotic disorders such as fibrotic diseases of the kidney, liver or lung or fibrotic cancers such as fibroadenomas, fibrosarcoas, etc. Any resistance of such fibrotic tissues to introduction of the gene transfer agent could be alleviated by treatment of the target cells with an agent that degrades the extracellular matrix-like material according to the methods of this invention.

Vectors useful in the methods of this invention include any gene delivery vehicle useful to deliver nucleic acids into a cell, in vitro or in vivo. When expression of the transgene is desired within the target cell, it is linked to expression control sequences within the vector. Examples of vectors are plasmids, viruses, such as adenoviruses, adeno-associated viruses (AAN), lentiviruses, herpes viruses, positive strand RΝA viruses, vaccinia viruses, baculoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.

The following example is for illustrative purposes only and is not intended to limit in any way the scope or applicability of the invention.

Example We first observed that cartilage explants are extremely difficult to infect with adenoviral vectors. However, we found that a two day incubation in culture media at 37 degrees C. sometimes resulted in a slight infection of the chondrocytes, mostly at the periphery of the tissue. Although promising, the consistency of this phenomenon was rather unpredictable. Furthermore, several days of incubation in culture media was clinically not significant since it did not provide a solution for in vivo gene transfer to the cartilage tissue. We did, however, note that the incubation was also associated with a loss of stainable proteoglycans. Accordingly, we hypothesized that the breakdown of the extracellular matrix of the cartilage explants was responsible for allowing the viral vector to penetrate the tissue.

We analyzed this process futher using an adenoviral vector referred to as Ad2/CMNβGAL-4. This vector is the same as the CMNβgal-1 vector described in D. Armentano et al, J. Virol., 71 :2408-2416 (1997), herein incorporated by reference in its entirey. This replication deficient adenoviral vector carries the gene for β-galactosidase. Accordingly, it is frequently used to monitor gene delivery. Specifically, the gene product that is expressed upon cell infection is easily detectable by adding the substrate of this enzyme, which turns X- GAL into a blue precipitate. We enzymatically treated bovine and human cartilage explants with hyaluronidase at concentrations of 0.5 and 1.0 % in F12 medium for one to three hours for the bovine cartilage and overnight for the human cartilage. The tissues were then infected with 10⁹ viral particles in small culture dishes and after 24-72 hours, the tissue was treated with X-GAL to reveal the efficiency of gene delivery. We found that increasing treatment with hyaluronidase resulted in increasing gene transfer efficiency. Where no or little staining was observed for non enymatically treated tissue, a substantial number of cells stained when treated with hyaluronidase.

To further confirm this observation the infected tissue was completely digested with collagenase to release the intact cells and the chondrocytes were plated on tissue culture plastic and allowed to grow as a monolayer. Again the cells were stained with X-GAL and blue cells were observed for both bovine and human cartilage, confirming that articular chondrocytes can be efficiently infected with adenoviral vectors directly in cartilage tissue as long as the extracellular matrix is degraded prior to gene transfer.

Claims

We Claim :

1. A method for increasing the efficiency of gene transfer into cells present in an extracellular matrix-like material comprising the steps of :

(a) treating the cells with an effective amount of an agent that degrades the extracellular matrix-like material under conditions sufficient to cause degradation of the extracellular matrix-like material; and

(b) subjecting the cells to gene transfer.

2. The method of claim 1, wherein the cells are subjected to gene transfer in the presence of the agent that degrades the extracellular matrix-like material.

3. The method of claim 1, wherein the agent that degrades the extracellular matrix-like material is removed prior to subjecting the cells to gene transfer.

4. The method of claim 1, wherein the cells are present in vitro.

5. The method of claim 1, wherein the cells are present in vivo.

6. A method for increasing the efficiency of gene transfer into chondrocytes present in an extracellular matrix-like material comprising the steps of :

(c) treating the chondrocytes with an effective amount of an agent that degrades the extracellular matrix-like material under conditions sufficient to cause degradation of the extracellular matrix-like material; and

(d) subjecting the cells to gene transfer.

7. The method of claim 6, wherein the chondrocytes are present in cartilage tissue in vivo.

8. The method of claim 6, wherein the chondrocytes are present in a cartilage explant in vitro.

9. The method of claim 6, wherein the gene transfer is accomplished with an adenoviral vector.

10. The use of an agent that degrades an extracellular matrix-like material for the formulation of a composition for the treatment of cells or tissues in order to increase the efficiency of gene transfer into said cells or tissues.

11. The use of claim 11 , wherein the composition additionally comprises a gene transfer agent.

12. A pharmaceutical composition comprising a gene transfer agent and an agent that degrades an extracellular matrix-like material.