Performing Department
School Of Veterinary Medicine
Non Technical Summary
Laminitis is such a common and devastating disease of horses that there is probably no one in the equine industry that still needs to be convinced of its importance. There are many forms of laminitis and presumed causes but some of the most disheartening cases are those associated with fractures and other major injuries. Many owners (and veterinarians) have suffered the heartbreak of losing a horse to laminitis of its opposite "good" foot while the original injury is being successfully treated. Unlike some other forms of laminitis, this is a situation in which a preventive approach could be taken because laminitis usually takes days to weeks to develop after the horse suffers the original injury. The underlying common mechanism of laminitis involves breakdown of the tissue that holds the coffin bone to the hoof wall. This tissue (the lamellae) is broken down by enzymes made within the hoof tissues or that circulate in the blood to the hoof. There are many potential causes for the release of these enzymes but the end result is the same - the hoof wall separates from the coffin bone and the foot becomes extremely painful for the horse. We propose that the technique of gene therapy can be used to prevent this breakdown. Gene therapy in this sense means the use of a virus to insert a gene into the cells of the hoof that will produce a protein to block the destruction of the hoof-coffin bone connection. This virus (Adeno-Associated Virus or AAV) is genetically modified to make it non-infectious. The virus can still take the desired gene into the horse's cells, but the virus cannot reproduce itself. Our laboratory has cloned the equine Tissue Inhibitor of MetalloProteinase -3 gene. The product of this gene, TIMP-3, is known to specifically block several of the most destructive enzymes that destroy the hoof-coffin bone connection. In this study, a common clinical technique, usually used for administering antibiotics in high concentrations to the lower limb, will be used to deliver the virus containing our desired gene. This should result in the therapeutic TIMP-3 being made in high enough concentrations to help prevent degradation of the hoof wall-coffin bone connection. While TIMP-3 is a logical choice to address the devastating degradation associated with laminitis, it is possible that TIMP-3 may not be the ultimate gene therapy "target". The extensive work being done now by other researchers exploring the mechanisms of the disease may identify more beneficial genes in the future, but this study should provide a strong basis for further investigations using gene therapy in equine laminitis. Importance to the Equine Industry: Laminitis is unquestionably one of the most devastating diseases of the horse. It affects all breeds of horses and results in intense suffering for horses afflicted with its most severe forms. The causes and mechanisms of laminitis are very complex but the final common result of laminitis is that there is separation of the coffin bone from the hoof wall and often unmanageable pain. Laminitis frequently will end a horse's competitive career and many horses are euthanized because of the persistent pain.
Animal Health Component
(N/A)
Research Effort Categories
Basic
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Applied
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Developmental
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Goals / Objectives
Disorganization of the epidermal lamellae, separation of basal cells and lysis of the basement membrane that occur in the pathology of most forms of laminitis are caused by uncontrolled, excessive Matrix Metalloproteinase (MMP) activation (Pollitt, 2008; Johnson et al. 1998; Pollitt et al. 1998). The filaments that attach lamellar basal cells to the basement membrane are MMP substrates and their degradation leads to failure of the attachment between hoof and distal phalanx. Coyne and colleagues (Coyne et al., 2008) demonstrated that gene expression of a Disintegrin and Metalloproteinase with Thromobospondin motifs gene (ADAMTS-4) was strongly upregulated in most horses with experimentally induced and naturally acquired laminitis and concluded that increased expression of ADAMTS-4 plays a central role in the pathophysiology of the disease. Treatments capable of inhibiting ADAMTS-4 may be able to prevent the development of laminitis if administered locally (Coyne et al., 2008). The most effective endogenous inhibitor of ADAMTS-4 is the Tissue Inhibitor of Metalloroteinase-3 (TIMP-3). Our specific long-term goal is to develop a gene therapy approach for the prevention laminitis in the contralateral foot of horses with major musculoskeletal injuries. We hypothesize that gene therapy can be used in vivo to protectively modify the internal tissue environment of the hoof lamellae without disrupting normal hoof architecture. For the proposed initial study, we hypothesize that 1) a recombinant adeno-associated viral vector (rAAV) can be used to transfer a therapeutic gene (TIMP-3) to equine lamellar tissue using local vascular perfusion and that 2) the transduction of a TIMP-3 vector into lamellar tissues will induce elevated TIMP-3 expression in the transduced tissue and will result in decreased MMP/ADAMTS activity. The following specific aims are designed to optimize a rAAV vector for gene delivery to equine lamellar tissues and to assess the effect of a rAAV-TIMP-3 transgene in equine lamellar tissues. Specific Aim 1. Determine the optimal recombinant AAV vector serotype for efficient transduction of equine lamellar tissue: Transduction efficiency of candidate rAAV-luciferase/LacZ transgene-containing vector serotypes will be assessed in monolayer culture of equine lamellar-derived cells and tissue explants. Specific Aim 2. Determine the biodistribution patterns of molecular markers of successful gene transfer (luciferase/LacZ) following regional intravenous perfusion with a rAAV vector into equine feet: Biodistribution of the injected rAAV-luciferase/LacZ transgene-containing vectors will be assessed utilizing real-time quantitative PCR, confocal and bioluminescent imaging of sectioned lamellar tissue. Aims 1 and 2 will be accomplished during the first year of the project. During the second year, specific aims 3 and 4 include: Specific Aim 3. Characterize levels of degradative enzymes (MMP/ADAMTS) and degradative enzyme inhibitor (TIMP-3) in rAAV-TIMP-3 vector-treated feet and in control (empty vector)-treated feet: The production of TIMP-3, MMP2, MMP 3, MMP 9, ADAMTS 4 and ADAMTS 5 will be evaluated in treated (rAAV-TIMP-3), co
Project Methods
Determine the optimal recombinant AAV vector serotype for efficient transduction of equine lamellar tissue. Transduction efficiency of candidate rAAV-luciferase/LacZ transgene-containing vector serotypes will be assessed in monolayer culture of equine lamellar-derived cells and tissue explants. Vector development will be based on the expertise of Dr. James Wilson and Dr. Julie Johnston at the University of Pennsylvania Vector Core facility. Initial vector design will be based on University of Pennsylvania Vector Core protocols (Bell et al., 2007). For comparisons of the relative efficiency of each vector to infect these tissues, Vector Core-developed rAAVs will be used to transduce in vitro cultures of lamellar cells and tissues. Lamellar cell and explant cultures will be established from samples collected from euthanized horses using cell and explant culture techniques developed previously in our laboratory (Davenport-Goodall et al., 2004). Initial vector selection will include rAAV1, 2, 5, 6.2, 7, 8, 9 and rh32.33. Vectors containing luciferase and LacZ reporter genes are currently available in our laboratories. For monolayer cell culture transduction experiments, vector dosage will be calculated in vector genome copies-per-cell (GC/cell) starting with 1x101 GC/cell up to 1x105 GC/cell with 1x104 cells/well in 96-well plates. Seven days post-transduction, cells will be washed, processed and analyzed for luciferase activity. Tissue explants will be 6mm2 tissue pieces cut from characteristic tissue. Culture medium for cell and explant culture will consist of DMEM:F12 containing 5% FBS, 1X antibiotic/antimycotic solution. All cultures will be maintained at 37 degrees centigrade in 5% carbon dioxide. Explants will be cultured in 96-well plates for transduction experiments. Vector dosage for explant experiments will be calculated in vector genome copies-per-culture well (GC/well) starting with 1x106 GC/well up to 1x1010 GC/well. Twenty-four hours post-transduction (and every 48-hours for 10 days thereafter), explants will be washed, processed and analyzed for luciferase activity. Luciferase activity will be recorded as counts (numerical photon data shown with a pseudocolor display) and flux (photons of light emitted per second per square centimeter per steradian), acquired by a charged cooled coupled device camera. The data relating to the transduced cells/explants will be compared against non-transduced cells/explants and a black background control. For a more qualitative assessment of the depth of vector penetration, additional explants may be transduced with a nuclear-targeted, lacZ-containing vector. Tissue sections will analyzed for depth of transgene tissue penetration from the bisected explant face. Transduced explants will be stained after bisection to ensure full-depth explant staining (Bell et al., 2007). Determine the biodistribution patterns of molecular markers of successful gene transfer (Luciferase/eGFP) following regional intravenous perfusion with a rAAV vector into equine feet. Biodistribution of the injected rAAV-luciferase/LacZ transgene-containing vectors will be assessed utilizing real-time quantitate