US 20090093867 A1
In accordance with the present invention, methods are provided for treating a patient through the use of ultraviolet light activated gene therapy. Embodiments of the present invention include methods for the utilization of light activated gene therapy to repair and/or rebuild damaged cartilage by introducing a desired gene into a patient's tissue.
76. A method of therapeutically increasing transduction of a viral vector comprising:
administering an adeno associated viral vector to a target cell in a subject; and
exposing the target cell to ultraviolet light having a wavelength from 320 nm to 400 nm, wherein the ultraviolet light is applied at a therapeutic dose, and wherein the dose is at least 500 J/m2, thereby therapeutically increasing transduction of the viral vector in the target cell.
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84. A method of promoting tissue repair, said method comprising:
administering an adeno associated viral vector to a target cell in a tissue, wherein said adeno associated viral vector comprises a gene that promotes tissue repair; and
exposing the target cell to a wavelength of ultraviolet light consisting of at least one wavelength of UVA light, thereby increasing transduction of the gene, wherein the target cell is exposed to a dose of at least 500 J/m2 of the at least one wavelength of UVA light.
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90. A method of increasing transduction of a viral vector without the use of a DNA damaging agent to drive said increase in transduction, said method comprising:
administering an adeno associated viral vector to a target cell; and
irradiating the target cell with at least one wavelength of ultraviolet light, wherein the wavelength of the ultraviolet light is selected from a wavelength consisting of a wavelength from 320 nm to 400 nm, thereby increasing transduction of the viral vector in the target cell without the use of a DNA damaging agent.
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The present application is a continuation of U.S. application Ser. No. 10/357,271, filed Jan. 31, 2003, the entirety of which is incorporated herein by reference. U.S. application Ser. No. 10/357,271, and the present application through U.S. application Ser. No. 10/357,271, claim the priority benefit under 35 U.S.C. §119(e) of Provisional Application No. 60/353,842, filed on Jan. 31, 2002. The present application is also related to Provisional Application No. 60/353,907, filed on Jan. 31, 2002, and U.S. application Ser. No. 10/357,273, filed on Jan. 31, 2003.
This invention was made with Government support under NIH Contract #AR45972, an RO1 grant awarded by NIAMS. The Government has certain rights in the invention.
1. Field of the Invention
The invention relates generally to the field of gene therapy. According to the present invention, devices and methods are provided for the combined use of light activated gene transduction (LAGT) employing ultraviolet light and recombinant adeno-associated virus (r-AAV) for the purpose of introducing a desired gene into a patient's tissue.
2. Description of the Related Art
Somatic cell gene therapy is a form of treatment in which the genetic material of a target cell is altered through the administration of nucleic acid, typically in the form of DNA. In pursuit of effective in vivo administration routes, scientists have harnessed the otherwise potentially deleterious ability of viruses to invade a target cell and “reprogram” the cell through the insertion of viral DNA. By encapsulating desirable genetic material in a viral particle, or “vector,” minus some of the viral DNA, the effective and targeted delivery of genetic material in vivo is possible. As applied to specific treatments, gene therapy offers the ability to adjust the expression of desirable molecules, including both intracellular and extracellular proteins, to bring about a desired biological result.
In particular, the desirable qualities of adeno-associated viruses (AAV) have led to further study of potential gene therapy uses. As a vehicle for gene therapy recombinant forms of AAV, or r-AAV, offer many advantages including the vector's ability to infect non-dividing cells (e.g., chondrocytes, cells within cartilage), the sustained target gene expression, the low immune response to the vector, and the ability to transduce a large variety of tissues. The AAV contains a single strand DNA (ssDNA) genome. Under normal conditions AAV is present in humans in a replication incompetent form, due to the fact the AAV alone does not encode the enzyme required for replication of the second DNA strand. Successful r-AAV transduction often requires the presence of a co-infection with an adenovirus or the exposure of the host cell to DNA damaging agents, such as γ-irradiation. The introduction of either the co-infection or the DNA damaging agents dramatically induces the rate limiting step of second strand synthesis, i.e. the second strand of DNA which is synthesized based on the vector inserted first strand. However, making use of these DNA damaging agents is impractical because the administration of an adenovirus co-infection to a patient is not practical or desirable and the site specific and safety issues involved with using γ-irradiation undesirable as well.
In the past, attempts have been made to induce r-AAV transduction in vitro using UV radiation having a wavelength of 254 nm. Unfortunately, no effective therapeutic method or apparatus was developed based on these experiments due to the long exposure times involved with using 254 nm UV radiation, the difficulties of delivering 254 nm UV radiation to a surgical target site, and the inability to position the 254 nm UV light source so as to allow effective penetration of a target cell.
Preferred embodiments of the present invention provide structures and methods for treating a patient using light activated gene therapy.
In accordance with an embodiment of the present invention, a method of introducing a desired gene into a patient's tissue is provided. The method includes locating a light probe proximate to the target cells. A long wavelength ultraviolet light is then transmitted through a light delivery cable to the light probe. Transduction of the ultraviolet light activated viral vector in target cells is activated using the light probe. An ultraviolet light activated viral vector is delivered proximate to target cells.
In accordance with another embodiment of the present invention, a gene therapy system for increasing the transduction of an ultraviolet activated viral vector is provided. The system includes a light source capable of long wavelength ultraviolet light output and a light probe configured to access an appropriate treatment site. An optical delivery cable is also provide to transmit the ultraviolet light from the light source to the light probe. In addition, light channeling optics are included to channel the light output into the light delivery cable.
In accordance with yet another embodiment of the present invention, a long wavelength ultraviolet radiation treatment system for increasing the transduction of an ultraviolet light activated viral vector in a patient's target cells is provided. The system includes a power source which powers a light source producing a long wavelength ultraviolet light beam. A timed shutter with a shutter control interface is included to selectively block the light beam. In addition, an optical coupler for channeling the light beam into a light delivery cable. A light probe, which is operatively connected to the light delivery cable, is configured to selectively output the light beam. Furthermore, the light probe is also configured to access the target cells.
In accordance with still another embodiment of the present invention, an implant system for introducing a desired gene into a patient's tissue is provided. The system includes an implant configured to be inserted into a patient's tissue in a minimally intrusive surgical procedure and an ultraviolet activated viral vector which is integrated with the implant.
A feature of certain preferred embodiments of this invention is the avoidance of the problems involved with using UV and γ-irradiation through the use of locally administered, long wavelength UV (i.e., greater than or equal to 255 nm) radiation in order to induce the target cell to more effectively stimulate the transduction of a UV activated viral vector, such as recombinant adeno-associated virus (r-AAV).
The term “AAV” refers to adeno-associated virus, while “r-AAV” refers to recombinant adeno-associated virus. Preferably, r-AAV includes only the desired gene to be introduced into the patient's tissue and the flanking AAV inverted terminal repeats (ITRs) that serve as the packaging signals.
“Ultraviolet radiation” and “ultraviolet light,” also known as “UV”, refer to the portions of the electromagnetic spectrum which have wavelengths shorter than visible light. The range of wavelengths considered to be ultraviolet radiation, from about 4 nanometers to about 400 nanometers, is further subdivided into three subgroups, UVA, UVB, and UVC. “UVA” is the portion of ultraviolet radiation which includes wavelengths from 320 nm up to and including 400 nm. “UVB” is the portion of ultraviolet radiation which includes wavelengths from 280 nm up to and including 320 nm. “UVC” is the portion of ultraviolet radiation having a wavelength less than 280 nm.
The term “long wavelength UV” refers to ultraviolet radiation or light having a wavelength equal to or greater than 255 nm, but not more than 400 nm.
A “viral vector” refers to a virus, or recombinant thereof, capable of encapsulating desirable genetic material and transferring and integrating the desirable genetic material into a target cell, thus enabling the effective and targeted delivery of genetic material both ex vivo and in vivo. A “UV activated viral vector” “UV light activated viral vector” is any virus, or recombinant thereof, whose replication is regulated by ultraviolet light. Recombinant adeno-associated virus (r-AAV) is included in the group of viruses labeled UV activated viral vectors. A “solid platform” is any structure designed to be inserted into the body for the purpose of aiding the treatment of the target site proximate to where the solid platform is inserted.
The term “LAGT” refers to light activated gene transduction, while “LAGT probe” or “light probe” or “long UV wavelength light probe” refers to the medical device which delivers long wavelength ultraviolet light to the target site and effectuates the transduction of the desired gene carried by the vector.
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Alternate embodiments employ as a light source, a lamp, such as a high intensity argon lamp. In these alternate embodiments, the UV radiation delivery system further includes a wavelength selecting device, such as a dichroic mirror and/or optical filter, set to transmit long wavelength UV and reject unwanted light wavelengths. In these embodiment, the wavelength selecting device and the dichroic mirror are preferably contained in the same housing as the light source.
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Surgery tools, other than injecting device shown in
It should be understood that structural support implants incorporating such conventional structures as, for example, but not limited to, plates, rods, wire, cables, hooks, screws, are also advantageously useful with preferred embodiments provided herein. The support structure may be formed from material such as, but not limited to, metal, carbon-fiber, plastic, and/or reabsorbable material.
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Embodiments of the present invention include both in vivo and ex vivo applications. In the ex vivo application the long wavelength UV light dose is applied to cells or biological material external to the patient and then delivered, preferably through injection, to the desired site of treatment. In the in vivo application the LAGT probe and the UV activated viral vector are preferably introduced to the treatment site using minimally invasive surgical techniques, such as stab incisions. Alternate in vivo embodiments employ direct visualization surgical techniques.
A UV activated viral vector is any virus, or recombinant thereof, whose replication is regulated by ultraviolet light. Preferred embodiments of UV activated viral vectors are viruses with single stranded DNA, the virus being capable of allowing a therapeutically significant increase in virus transduction when a virus infected target cell is exposed to a therapeutic does of ultraviolet radiation. More preferred embodiments include UV activated viral vectors capable of infecting non-dividing cells, effectuating sustained target gene expression, eliciting a low immune response to the vector, and possessing an ability to transduce a large variety of tissues.
Proof of principle experiments, both ex vivo and in vivo based, are currently under way and can determine the optimal wavelengths for activating the gene therapy. The determination of more preferred wavelengths is based on among other factors, the ability to effectively penetrate a target cell, ease and efficiency of fiber optic transmission, the ability to trigger r-AAV transduction, and the length of time a patient must be exposed to receive a therapeutic dose of ultraviolet radiation. Preferably, the LAGT system delivers long wavelength ultraviolet radiation in the range of 315 nm to 400 nm. Current experiments support the use of ultraviolet radiation having a wavelength from 315 nm to 355 nm, more particularly about 325 nm, but it is believed that these experiments will ultimately support ultraviolet radiation having a wavelength from 315 nm to 400 nm. In addition, alternate embodiments employ a laser which produces ultraviolet radiation having a wavelength of about 290 nm. Once specific wavelengths are determined, the disclosed components can be optimized for these specific wavelengths.
The wavelength of the ultraviolet light generated in order to activate UV activated viral vector transduction, including r-AAV transduction, in target cells is preferably 255, 256, 258, 265, 275, 285, 290, 295, 305, 314, 325, 335, 345, 355, 365, 375, 385, 395, or 400 nanometers. More preferably, the wavelength of the ultraviolet light is 290, 295, 300, 305, 310, 315, 316, 317, 322, 325, 327, 332, 337, 342, 347, 352, 357, 362, 367, 372, 377, 382, 387, 392, 393, 394, 395, 396, 397, 398, or 399 nanometers. Most preferably, the wavelength of the ultraviolet light is 325 nanometers.
Tables 1-3 are charts of example growth factors, signaling molecules and/or transcription factors which desired genes, selected based on the desired use (e.g., implant integrated vs. in solution) and outcome (e.g., osteo-integration, spine fusion, perioprosthetic osteolysis, and/or cartilage repair/regeneration) inserted into a UV activated viral vector could encode for. The lists contained in Tables 1-3 are provided for illustrative purposes and should not be taken as limiting the embodiments of the invention in any way.
The results of a completed proof of principle experiment are shown below in Example 1.
A. Isolation of Human Mesenchymal Stem Cells
Human Mesenchymal Stem Cells (hMSC) were isolated from patient blood samples harvested from the iliac crest. The blood samples were diluted in an equal volume of sterile Phosphate Buffered Saline (PBS). The diluted sample was then gently layered over 10 ml of Lymphoprep (Media Prep) in a 50 ml conical tube (Corning). The samples were then centrifuged at 1800 rpm for 30 minutes. This isolation protocol is a standard laboratory technique, and the resulting gradient that formed enabled the isolation of the hMSCs from the layer immediately above the Lymphoprep. The isolated fraction was placed into a new 50 ml conical tube, along with an additional 20 ml of sterile PBS. The sample was centrifuged at 1400 rpm for 8 minutes. The supernatant was removed the cell pellet was resuspended in 20 ml for fresh PBS, and centrifuged again for 8 minutes at 1400 rpm. Afterwards the supernatant was removed, the cell pellet was resuspended in 10 ml of Dulbecco's Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin (P/S) (Invitrogen). The hMSCs were grown and passed as necessary in a 37°/5% CO2, water jacketed incubator (Form a Scientific).
B. 325 nm UV treatment of Human Mesenchymal Stem Cells
Prior to irradiation, hMSCs were plated at a density of 5×104 cells/well in 12-well plates. The cells were allowed to sit down overnight. The next morning the media was removed immediately prior to irradiation. The cells were irradiated at various doses (500 J/m2, 1000 J/m2, 3000 J/m2, 6000 J/m2, or 10,000 J/m2) of 325 nm UV light using a helium-cadmium laser system (Melles Griot). After irradiation, fresh media, either with or without recombinant adeno-associated virus was added to the wells.
C. Infection of Human Mesenchymal Stem Cells with Recombinant Adeno-Associated Virus
Infections were carried out in 12-well dishes. The cells were infected at various multiplicities of infection (MOIs=10, 100, and 1000), using a recombinant adeno-associated virus carrying the bacterial β-galactosidase reporter gene (rAAV-LacZ via UNC-Chapel Hill Gene. Therapy Vector Core Facility). After being irradiated, the cells were infected with the predetermined amount of virus in a total volume of 500 μl of DMEM/10% FBS/1% P/S. Two hours after the initial infection, and additional 1 ml of media was added to the cultures. The cultures were then allowed to incubate (37°/5% CO2) for forty-eight hours before harvest for analysis.
D. Quantifying Recombinant Gene Expression
Forty-eight hours after infection, the cells were harvested; cell lysates were made and analyzed using a commercially available Luminescent β-gal Reporter System. (BD Biosciences), Briefly, experimental cell samples were removed from the 12-well dish using 0.25% Trypsin-EDTA. The cell suspension was transferred to a 1.5 ml conical tube and the cells were pelleted via a 15 second centrifugation at 13,000 rpm. The cell pellet was washed using two successive rounds of resuspension in ice cold PBS and pelleting for 15 seconds at 13,000 rpm. The final pellet was resuspended in 75 μl of Lysis Buffer (100 mM K2HPO4, 100 mM KH2PO4, 1 M DTT) and subjected to three rounds of freeze/thaw in an isopropanol dry ice bath and a 37° water bath. The lysates were centrifuged for a final time for 5 minutes at 13,000 rpm. Aliquots (15 μl) of the resulting supernatant were incubated with the provided substrate/buffer solution for one hour and then analyzed using a standard tube luminometer. The read out of this analysis is expressed in Relative Light Units (RLU) in the Results section.
A. Exposure to 325 Nm UV Increased the Level of Reporter Gene Expression
Exposure to 325 nm UV prior to infection with rAAV-LacZ had a dose dependent increase in LacZ reporter gene expression at each of the MOI's used. The controls for each experiment were as follows: Mock (cells alone, no treatment) and cells treated with each of the various UV dosages (500 J/m2, 1000 J/m2, 3000 J/m2, 6000 J/m2, which had RLU levels consistent with the Mock cultures (data not shown). Statistical significance was calculated using the Student T-Test. The results are shown in
Although this invention has been disclosed in the context of certain preferred embodiments and an Example, it will be understood by those skilled in the art that the present invention extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses of the invention and obvious modifications thereof. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the invention may be practiced in many ways. Thus, it is intended that the scope of the present invention herein disclosed should not be limited by the particular disclosed embodiments described above, but should be determined only by a fair reading of the claims that follow and any equivalents thereof.