Growth Factor Gene Transfer Research Articles

Saturday, November 2, 2002 2:14–2:57 pm Concurrent Session 6A: Bone Morphogenic Protein: 2:14 Bone morphogenic protein–2 facilitates expression of chondrogenic not osteogenic phenotype in human intervertebral disc cells

Purpose of study: With the introduction of growth factors and development of vector technology, it is possible to genetically modify intervertebral disc cells by transfer of growth factor genes to upregulate matrix synthesis. Bone morphogenic protein (BMP)-2 was known to play a crucial role in osteoblast differentiation and bone formation. The bone-forming potential of BMP-2 raises the research question, Can BMP-2 upregulate expression of chondrogenic phenotype rather than osteogenic phenotype in the intervertebral disc? Therefore, the objective of this in vitro study is to determine the effect of recombinant human BMP-2 (rhBMP-2) on proteoglycan synthesis and mRNA expression of matrix components, that is, aggrecan, collagen type 1, collagen type 2 and osteocalcin in human intervertebral disc cells.Methods used: We harvested cervical and lumbar disc tissue from six patients during surgical disc procedures. The disc cells were isolated from disc tissue by sequential enzymatic digestion and cultured. Then cultures were incorporated into alginate beads as described. rhBMP-2 (R&D) was administered to the cultures with various concentration. Newly synthesized proteoglycan was assessed by 35S incorporation using chromatography on Sephadex G-25 in PD-10 column. Reverse transcriptase polymerase chain reaction (RT-PCR) for expression of aggrecan, collagen type 1, collagen type 2 and osteocalcin mRNA was performed.of findings: In the rhBMP-2–treated cultures, there was increased newly synthesized proteoglycan (1.6-fold in 300 ng/ml, 3.5-fold in 1,500 ng/ml of rhBMP-2) and increased expression of aggrecan, collagen type 1 and collagen type 2 mRNA compared with untreated control in a dose-dependent manner. However, rhBMP-2 did not upregulate expression of osteocalcin mRNA in a given dose.Relationship between findings and existing knowledge: The result of this study demonstrated that the effect of BMP-2 on human intervertebral disc cells is chondrogenic rather than osteogenic. Also BMP-2 upregulates synthesis of matrix as proved by proteoglycan synthesis, mRNA expression of collagen 1, 2 and aggrecan.Overall significance of findings: In human intervertebral disc cells, rhBMP-2 clearly upregulates mRNA expression of chondrogenic component, that is, aggrecan, collagen type 1 and collagen type 2 without upregulating expression of mRNA of osteocalcin with given dose. The effect of rhBMP-2 was dose dependent in proteoglycan synthesis and mRNA expression of aggrecan, collagen type 1 and collagen type 2. In conclusion, this study raises the possibility that rhBMP-2 and BMP-2 encoding gene can be anabolic agents for regenerating matrix of the intervertebral disc.Disclosures: BMP-2: Investigational.Conflict of interest: Hwan-Mo Lee, grant research support, Brain Korea 21 Project (government).

Gene transfer of hepatocyte growth factor to subarachnoid space in cerebral hypoperfusion model.

Although cerebral hypoperfusion caused by cerebral occlusive disease leads to cerebral ischemic events, an effective treatment has not yet been established. Recently, a novel therapeutic strategy for ischemic disease using angiogenic growth factors to expedite and/or augment collateral artery development has been proposed. Therapeutic angiogenesis might be useful for the treatment of cerebral occlusive disease. Hepatocyte growth factor (HGF) is a potent angiogenic factor, in addition to vascular endothelial growth factor (VEGF), whereas in the nervous system HGF also acts as neurotrophic factor. Therefore, we hypothesized that gene transfer of these angiogenic growth factors could induce angiogenesis, thus providing an effective therapy for cerebral hypoperfusion or stroke. In this study, we employed a highly efficient gene transfer method, the viral envelop (Hemagglutinating Virus of Japan [HVJ]-liposome) method, because we previously documented that beta-galactosidase gene could be transfected into the brain by the HVJ-liposome method. Indeed, we confirmed wide distribution of transgene expression using beta-galactosidase via injection into the subarachnoid space. Of importance, transfection of HGF or VEGF gene into the subarachnoid space 7 days before occlusion induced angiogenesis on the brain surface as assessed by alkaline phosphatase staining (P<0.01). In addition, significant improvement of cerebral blood flow (CBF) was observed by laser Doppler imaging (LDI) 7 days after occlusion (P<0.01). Unexpectedly, transfection of HGF or VEGF gene into the subarachnoid space immediately after occlusion of the bilateral carotid arteries also induced angiogenesis on the brain surface and had a significant protective effect on the impairment of CBF by carotid occlusion (P<0.01). Interestingly, coinjection of recombinant HGF with HGF gene transfer revealed a further increase in CBF (P<0.01). Here, we demonstrated successful therapeutic angiogenesis using HGF or VEGF gene transfer into the subarachnoid space to improve cerebral hypoperfusion, thus providing a new therapeutic strategy for cerebral ischemic disease.

1102. Lung Permeability Induced by Adenovirus-Mediated Vascular Endothelial Growth Factor (VEGF) Gene Transfer Is Mediated Via Direct Endothelial Activation by VEGF

1102. Lung Permeability Induced by Adenovirus-Mediated Vascular Endothelial Growth Factor (VEGF) Gene Transfer Is Mediated Via Direct Endothelial Activation by VEGF

721. Preliminary Clinical Results from TREAT-HGF (Japan Trial To Treat Peripheral Arterial Disease by Therapeutic Angiogenesis Using Hepatocyte Growth Factor Gene Transfer)

721. Preliminary Clinical Results from TREAT-HGF (Japan Trial To Treat Peripheral Arterial Disease by Therapeutic Angiogenesis Using Hepatocyte Growth Factor Gene Transfer)

Angiogenic gene therapy for experimental critical limb ischemia: acceleration of limb loss by overexpression of vascular endothelial growth factor 165 but not of fibroblast growth factor-2.

Recent studies suggest the possible therapeutic effect of intramuscular vascular endothelial growth factor (VEGF) gene transfer in individuals with critical limb ischemia. Little information, however, is available regarding (1) the required expression level of VEGF for therapeutic effect, (2) the related expression of endogenous angiogenic factors, including fibroblast growth factor-2 (FGF-2), and (3) the related adverse effects due to overexpression of VEGF. To address these issues, we tested effects of overexpression of VEGF165 using recombinant Sendai virus (SeV), as directly compared with FGF-2 gene transfer. Intramuscular injection of SeV strongly boosted FGF-2, resulting in significant therapeutic effects for limb salvage with increased blood perfusion associated with enhanced endogenous VEGF expression in murine models of critical limb ischemia. In contrast, VEGF165 overexpression, 5-times higher than that of baseline on day 1, also strongly evoked endogenous VEGF in muscles, resulting in an accelerated limb amputation without recovery of blood perfusion. Interestingly, viable skeletal muscles of either VEGF165- or FGF-2-treated ischemic limbs showed similar platelet-endothelial cell adhesion molecule-1-positive vessel densities. Maturation of newly formed vessels suggested by smooth muscle cell actin-positive cell lining, however, was significantly disturbed in muscles with VEGF. Further, therapeutic effects of FGF-2 were completely diminished by anti-VEGF neutralizing antibody in vivo, thus indicating that endogenous VEGF does contribute to the effect of FGF-2. These results suggest that VEGF is necessary, but should be delicately regulated to lower expression to treat ischemic limb. The therapeutic effect of FGF-2, associated with the harmonized angiogenic effects seen with endogenous VEGF, provides important insights into therapeutic angiogenesis.

Gene Transfer of Naked VEGF Plasmid Induces the Formation of Microvessels but not Mature Collaterals in Ischaemic Limb Muscles

Background: Several studies have demonstrated increased numbers of angiographically detectable collaterals after vascular endothelial growth factor (VEGF) gene transfer. However, VEGF appears to be insufficient for stimulating the growth of mature blood vessels. Therefore, we decided to reinvestigate in what way the VEGF gene transfer to rabbit ischaemic muscle can restore blood flow impaired by femoral artery excision.

Gene therapy concepts for promoting wound healing

Growth factors are important players in orchestrating all stages of wound healing. They are mitogen, chemotactic, they regulate cell-cell interactions and influence synthesis and composition of extra-cellular matrix components. The use of growth factors to stimulate wound healing is a promising new therapeutic approach to repair chronic tissue defects. The delivery of genetic material offers an attractive treatment modality to produce an appropriate amount of growth factor proteins within the wound site. This review discusses several methods of growth factor gene transfer into wound tissue and presents recent experimental results of gene therapy approaches in stimulating wound repair.

Attenuated acute liver injury in mice by naked hepatocyte growth factor gene transfer into skeletal muscle with electroporation.

Hepatocyte growth factor (HGF) plays an essential role in hepatic development and regeneration, and shows proliferative and antiapoptotic activity in hepatocytes. To establish an effective new method for HGF gene transfer in vivo and to investigate its effects in acute experimental liver injury. Eight week old female mice were used. Rat HGF gene in a modified pKSCX plasmid was transferred to the tibialis anterior muscle by electroporation using a pulse generator. Four days later, plasma HGF concentrations were determined by enzyme linked immunosorbent assay every two days for three weeks. To confirm the efficacy of electroporation, a plasmid bearing green fluorescence protein (GFP) was transferred similarly. Four days after electroporation, carbon tetrachloride (CCl(4)) was administered to mice to induce acute liver injury. Plasma alanine aminotransferase (ALT) activity was measured. Hepatic apoptosis was assessed by Hoechst 33258 staining and the TUNEL method. Fluorescence microscopy showed strong green fluorescence where the GFP gene had been transferred into muscle. In mice given the HGF gene, HGF in plasma was increased up to fourfold from pretreatment amounts, peaking 6-9 days after electroporation and quickly decreasing within three weeks. Compared with the group without HGF transfer, the percentage of apoptotic hepatocytes after CCl(4) intoxication was significantly lower, as was ALT activity. In addition, ALT activity normalised more rapidly in the HGF gene transfer group. Naked DNA injection and transfer by electroporation efficiently brings about HGF expression in vivo, which can attenuate acute liver injury.

Genetic engineering of meniscal allografts.

Allograft meniscal transplantation represents one of the few available treatment options after menisectomy. Despite acceptable early results, a considerable controversy exists with regard to poor graft regeneration, shrinkage and biomechanical failure of transplanted menisci. Transfer of specific growth factor genes may improve the regeneration process of meniscal allografts. The aim of this study was to investigate the feasibility of gene transfer in meniscal allografts in rabbits. Four different viral vectors encoding marker genes, including lacZ, luciferase, and green fluorescence protein were used to investigate viral transduction in 50 lapine menisci for 4 weeks in vitro. Subsequently, 16 unilateral meniscus replacements were performed with ex vivo retrovirally transduced meniscal allografts, and the expression of the lacZ gene was examined histologically at 2, 4, 6, and 8 weeks after transplantation. Gene expression in the superficial cell layers of the menisci can be detected for up to 4 weeks in vitro, but the level of gene transfer declined over time. The transduction with retrovirus showed better persistence and deep penetration of the menisci with infected cells. In vivo, declining numbers of beta-galactosidase-positive cells were also detected in retrovirally transduced allografts up to 8 weeks. Consistently, transduced cells were found at the menisco-synovial junction of the transplants and in deeper layers of the menisci. There was no evidence of cellular immune response in the transduced transplants. This investigation showed a prospective for growth factor delivery in auto- and allografts. In further experiments, vectors expressing therapeutic proteins such as growth factors will be investigated to assess their potential to improve remodeling and healing of meniscal allografts.

Therapeutic success and efficacy of nonviral liposomal cDNA gene transfer to the skin in vivo is dose dependent.

It is well documented that responses to growth factor treatment typically display bell-shaped dose responses that can significantly affect efficacy. Here we tested the hypothesis that nonviral liposomal gene delivery also displays this characteristic. We chose two different growth factors, keratinocyte growth factor (KGF) and insulin-like growth factor-I (IGF-I) CMV-driven transfecting constructs at three different concentrations and assessed efficacy on several physiological parameters that are descriptive of wound healing progress in a burn-wound healing model. Rats were given a 60% TBSA scald burn and randomly divided into one of seven groups to receive weekly subcutaneous injections of liposomes containing the cDNA for KGF (0.2 microg, 2.2 microg, or 22.2 microg), or liposomes containing the cDNA for IGF-I (0.2 microg, 2.2 microg, or 22.2 microg) at various concentrations, but constant liposome:DNA ratios and a LacZ gene (0.2 microg) CMV-driven construct for beta-galactosidase as vehicle and marker gene. Transfection was confirmed by histology for beta-galactosidase. Physiological efficacy was evaluated by measuring the wound healing parameters that define dermal and epidermal regeneration. Transfection products were found in the cytoplasm of rapidly dividing cells of the granulation tissue. Different doses of the nonviral cDNA gene transfer coding for KGF or IGF-I resulted in different outcomes for dermal and epidermal regeneration. There was a dose-dependent response to both growth factor gene transfers that was not dissimilar from that typically displayed by treatment with growth factor proteins. Both concentrations below and above the optimal concentration of DNA:liposomal preparations did not yield the results observed at the optimal concentration.

Overexpression of human insulin-like growth factor-I promotes new tissue formation in an ex vivo model of articular chondrocyte transplantation.

Articular cartilage, the tissue that forms the gliding surface of joints, has a poor regenerative capacity. Insulin-like growth factor-I (IGF-I) is a polypeptide that is anabolic and mitogenic for cartilage. Transfection of articular chondrocytes with an expression plasmid vector containing the cDNA for human IGF-I under the control of the cytomegalovirus promoter/enhancer led to expression of the transgene and synthesis of biologically relevant amounts of IGF-I protein. Transplantation of transfected articular chondrocytes on to the surface of articular cartilage explants led to the formation of a new tissue layer on the cartilage explant surface. The new tissue was characterized by the presence of type II collagen and proteoglycan and by the absence of type I collagen, consistent with hyaline-like cartilage. The tissue formed by the chondrocytes expressing IGF-I was thicker and contained more cells than controls transfected with an expression plasmid vector containing the Escherichia coli (E. coli) beta-galactosidase (lacZ) gene. Transplantation of articular chondrocytes that overexpress human IGF-I also increased DNA synthesis and the synthesis of glycosaminoglycans by the underlying explant cartilage chondrocytes. These results identify a mechanism by which IGF-I may simultaneously promote chondrogenesis and shift cartilage homeostasis in an anabolic direction. The data further suggest that therapeutic growth factor gene transfer may be applicable to articular cartilage.

Assessment of risks associated with cardiovascular gene therapy in human subjects.

Clinical trials of cardiovascular gene therapy, whether using viral (53%) or nonviral (47%) vectors, have thus far disclosed no evidence indicative of inflammatory or other complications, including death, directly attributable to the vector used. Indeed, despite the fact that initial trials of cardiovascular gene therapy targeted patients with end-stage vascular disease, including critical limb ischemia and refractory myocardial ischemia, the mortality for patients enrolled in clinical trials of cardiovascular gene therapy reported to date compares favorably with mortality for similar groups of patients in contemporary controlled studies of medical or interventional therapies. The most common morbidity reported after cardiovascular gene transfer is lower extremity edema; in contrast to data involving genetically engineered mice, however, evidence of life- or limb-threatening edema has not been described in any patients, including patients after gene transfer for myocardial ischemia. Concerns regarding the potential for angiogenic cytokines to promote the progression of atherosclerosis are not supported by angiographic follow-up of patients with coronary or peripheral vascular disease. The levels and duration of gene expression investigated for therapeutic angiogenesis transfer have been unassociated with hemangioma formation. Likewise, there is little evidence from either preclinical or clinical studies to support the notion that the administration of angiogenic growth factors, per se, is sufficient to stimulate the growth of neoplasms. Patients enrolled in clinical studies of angiogenic cytokines, including patients with diabetes and a previous history of retinopathy, have disclosed no evidence to suggest that ocular pathology is a risk of angiogenic growth factor gene transfer.

Gene therapy for therapeutic angiogenesis in critically ischaemic lower limb – on the way to the clinic

Currently, no effective pharmacological treatment is available for vascularisation defects in lower limbs. Many patients presenting with persistent pain and ischaemic ulcers are not suitable candidates for surgical or endovascular approaches. Further refinement of the available methods will undoubtedly lead to a more active approach towards treatment of peripheral arterial occlusive disease (PAOD). Recently, therapeutic angiogenesis, in the form of recombinant growth factor administration or gene therapy, has emerged as a novel tool to treat these patients. However, improved gene transfer methods and better understanding of blood vessel formation are required to bring therapeutic angiogenesis to clinical practice. Here we review the clinical problem (PAOD), mechanisms of blood vessel formation (angiogenesis, vasculogenesis and arteriogenesis), experimental evidence and clinical trials for therapeutic angiogenesis in critically ischaemic lower limbs. Also, angiogenic growth factors, including vascular endothelial growth factors (VEGFs) and fibroblast growth factors (FGFs), delivery methods, and vectors for gene transfer in skeletal muscle, are discussed. In addition to vascular growth, gene transfer of growth factors may enhance regeneration, survival, and innervation of ischaemic skeletal muscle. Nitric oxide (NO) appears to be a key mediator in vascular homeostasis and growth, and a reduction in its production by age, hypercholesterolemia or diabetes leads to the impairment of ischaemic disorders.

A Novel Recombinant Basic Fibroblast Growth Factor and Its Secretion

Basic fibroblast growth factor (FGF-2) is a pleiotropic mitogen which plays an important role in cell growth, differentiation, migration, and survival in different cells and organ systems. Recently, several clinical applications for FGF-2 gene transfer are being evaluated in wound healing and collateral artery development to relieve myocardial and peripheral ischemia due to the ability of FGF-2 to regulate the growth and function of vascular cells. However, FGF-2 lacks a classical hydrophobic secretion signal peptide, the FGF-2 chimeras containing various signal sequences have been explored. In this study, a novel recombinant 4sFGF-2 was constructed by replacing nine residues from the amino-terminus of native FGF-2 (Met1 to Leu9) with eight amino acid residues of signal peptide of FGF-4 (Met1 to Ala8) to better increase the secretion level of FGF-2. When the recombinant FGF-2 gene, cloned into the expression vector with CMV promoter, was expressed in COS-7 cells, the recombinant 4sFGF-2 was highly secreted into the media. The secreted 4sFGF-2 showed the same biological activity as the native FGF-2 in the dose-response effects on DNA synthesis and cell growth of rat aortic smooth muscle cells (RASMCs) and NIH3T3 cells. The 4sFGF-2 also was able to activate MAPK as wild FGF-2 in RASMCs. These results indicate that a novel recombinant 4sFGF-2 may be useful as clinical applicability of angiogenic growth factor gene transfer.

Gene transfer and expression of platelet-derived growth factors modulate periodontal cellular activity.

Platelet-derived growth factor (PDGF) is a potent stimulator of wound healing. PDGF gene therapy may promote greater periodontal regeneration than local protein application, due to sustained growth factor delivery to the target tissue. This investigation tested the ability of recombinant adenoviruses (rAds) encoding PDGF-A or PDGF-1308 (a PDGF-A dominant-negative mutant that disrupts endogenous PDGF bioactivity) to affect cells derived from the periodontium. Osteoblasts, periodontal ligament fibroblasts, and gingival fibroblasts were transduced with rAds, and gene expression, DNA synthesis, and cell proliferation were evaluated. The results revealed strong message for the PDGF-A gene for 7 days following gene delivery. Ad2/PDGF-A enhanced the mitogenic and proliferative response in all cell types, while Ad2/PDGF-1308 potently inhibited mitogenesis and proliferation. In conclusion, Ad2/PDGF can effectively transduce cells derived from the periodontium and promote biological activity equivalent to PDGF-AA. These studies support the potential use of gene therapy for sustained PDGF release in periodontal tissues.

Matrix Synthesis of Human Intervertebral Disc Cells According to Grade of Degeneration:Under the Basal State and TGF-β1 Stimulation

Study Design : In vitro experiment to determine the matrix synthesis of human intervertebral disc (IVD) cell according to grade of degeneration. Objectives : To quantify proteoglycan synthesis of human IVD cells in various grade of degeneration under the stimulation of T G F -β1 . Summary of Literature Review : Sophisticated method to delivery of growth factors, in continuous manner, is the genetic modification of disc cells through gene transfer. Poor responsiveness of degenerated disc to anabolic stimuli can mitigate potential application of growth factor or therapeutic gene transfer in the management of degenerative disc disease Materials and Methods : IVD tissue was obtained from nineteen patients during surgical disc procedure. Grade of degeneration was confirmed by preoperative MRI. Isolation and three dimensional culture of disc cells were performed. Disc cells were treated with exogenous TGF-β1. Newly synthesized proteoglycans were assessed by S-sulfate incorporation using chromatography on Sepadex G-25 in PD-10 columns. One-way analysis of variance with Fishers protected LSD post-hoc test was performed to compare amount of newly synthesized proteoglycans and power analysis was also conducted. Significance level was set p<0.05. Results : Difference between cultures of control (grade I) and degenerated (grade II-V) discs in proteoglycan synthesis in basal condition was statistically insignificant (p=0.35, power=0.21-0.63). Difference between cultures of control and degenerated disc in proteoglycan synthesis under the stimulated condition with TGF-β1 was also statistically insignificant (p=0.54, power=0.240.47). However cultures in stimulated condition showed increased amount of newly synthesized proteoglycans compared to those of basal condition regardless of the grade of degeneration (p<0.05). Conclusion : Anabolic response of human intervertebral disc cells is relatively insensitive to grade of disc degeneration, which facilitate application of gene therapy in various conditions of disc degeneration.

Adeno-associated viral vector-mediated vascular endothelial growth factor gene transfer induces neovascular formation in ischemic heart.

Vascular endothelial growth factor (VEGF) plays important roles in physiological and pathological angiogenesis. Recent studies have demonstrated that direct injection of VEGF protein, plasmid DNA, or an adenoviral vector encoding the VEGF gene into ischemic myocardium or limb can induce collateral blood vessel formation and improve perfusion of the ischemic areas. However, these approaches have limitations ranging from a short-lasting effect to angioma formation. In this study, we investigated the feasibility of using adeno-associated viral (AAV) vectors to deliver VEGF genes to mouse myocardium. A cytomegalovirus promoter was used to drive genes for a human VEGF isoform, VEGF(165), and LacZ. A mouse myocardial ischemic model was generated by ligation of the anterior descending coronary artery. Approximately 10(11) copies of the AAV-VEGF vector mixed with 10(10) copies of AAV-LacZ were injected to one site of normal myocardium and a total of 10(11) copies of AAV-VEGF were injected to multiple sites of myocardium around the ischemic region. LacZ gene expression was observed up to 3 months after the vector inoculation. After AAV-VEGF inoculation, neoangiogenesis was observed in the ischemic heart model but not in normal heart tissue. An inflammatory-cell infiltration was not observed in the AAV-VEGF- and AAV-LacZ-inoculated hearts, and angioma-like structure was not observed. These results indicated that injection of the AAV vector directly to myocardium could mediate efficient gene transfer and transgene expression and that VEGF gene delivered by AAV vector can induce angiogenesis in ischemic myocardium.

The status of gene therapy in cardiovascular medicine

Vascular gene transfer potentially offers new treatments for cardiovascular diseases. It may be used to overexpress therapeutically important proteins and correct genetic defects, and to test experimentally the effects of various genes in a local vascular compartment. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) gene transfers have improved blood flow and collateral development in ischemic limb and myocardium. Promising therapeutic effects have been obtained in animal models of restenosis or vein-graft thickening with the transfer of genes coding for VEGF, nitric-oxide synthase, thymidine kinase, retinoblastoma, growth arrest homoeobox, tissue inhibitor of metalloproteinases, cyclin or cyclin-dependent kinase inhibitors, fas ligand and hirudin, and antisense oligonucleotides against transcription factors or cell-cycle regulatory proteins. First experiences of VEGF gene transfer and decoy oligonucleotides in human beings have been reported. However, further developments in gene transfer vectors, gene delivery techniques and identification of effective treatment genes will be required before the full therapeutic potential of gene therapy in cardiovascular disease can be assessed.

Human intervertebral disc cells are genetically modifiable by adenovirus-mediated gene transfer: implications for the clinical management of intervertebral disc disorders.

Human intervertebral disc cells were cultured in monolayer and treated with adenovirus-containing marker genes to determine the susceptibility of the cells to adenovirus-mediated gene transfer. To test the efficacy of the adenovirus-mediated gene transfer technique for transferring exogenous genes to human intervertebral disc cells in vitro. Upregulated proteoglycan synthesis after direct in vivo adenovirus-mediated transfer of growth factor genes to the rabbit intervertebral disc has previously been reported. Before contemplating extending this approach to the treatment of human disc disease, it is necessary to demonstrate that human intervertebral disc cells are indeed susceptible to adenovirus-mediated gene transduction. Human intervertebral disc cells were isolated from disc tissue obtained from 15 patients during surgical disc procedures. The cells were cultured in monolayer and treated with saline containing five different doses of adenovirus carrying the lacZ gene (Ad/CMV-lacZ), saline containing adenovirus carrying the luciferase gene (Ad/CMV-luciferase), or saline alone. Transgene expression was analyzed by 5-bromo-4-chloro-3-indolyl-beta-galactosidase (X-Gal) staining and luciferase assay. Adenovirus efficiently transferred lacZ and luciferase marker genes to cells from degenerated discs as well as to cells from nondegenerated discs. A minimum dose of 150 MOI Ad/CMV-lacZ was found to be sufficient to achieve transduction of approximately 100% of disc cells-regardless of patient age, sex, surgical indication, disc level, and degeneration grade. No statistically significant difference in the luciferase activities could be detected in disc cell cultures from degenerated and nondegenerated discs treated with Ad/CMV-luciferase. In vitro transducibility of human intervertebral disc cells by adenovirus is relatively insensitive to disc degeneration grade. Because the rate-limiting step for successful gene therapy is the ability to transfer genes efficiently to the target tissue, the achievement of efficient gene transfer to human intervertebral disc cells(using a direct, adenovirus-mediated approach) is an important and necessary step in the development of gene therapy strategies for the management of human intervertebral disc disorders.

Insulinlike growth factor-I gene therapy applications for cartilage repair.

Cartilage function after resurfacing with cell-based transplantation procedures or during the early stages of arthritic disease may be bolstered by the addition of growth factor genes to the transplanted tissue. Insulinlike growth factor-I maintains chondrocyte metabolism in normal cartilage homeostasis and has been shown to improve cartilage healing in vivo. Given the relatively short half-life of insulinlike growth factor-I in biologic systems, however, maintenance of effective concentrations of this peptide has necessitated either very high initial doses or repeated treatment. Delivery of the insulinlike growth factor-I gene, using a deleted adenovirus vector, specifically targeting graftable articular chondrocytes, bone marrow-derived chondroprogenitor cells, or synovial lining cells, may provide more durable insulinlike growth factor-I fluxes to articular tissues. Cultured equine articular chondrocytes, mesenchymal stem cells, synovial explants, and synovial intimal cells were readily transfected with an E1-deleted adenoviral vector containing equine insulinlike growth factor-I coding sequence. Optimal viral concentrations for effective transduction were 100 multiplicities of infection in synoviocytes, 500 multiplicities of infection in chondrocytes, and 1000 multiplicities of infection in mesenchymal stem cells. Production of insulinlike growth factor-I ligand varied from 65 ng/mL to 246 ng/mL in medium from chondrocytes and synovial explants, respectively. For chondrocytes, these concentrations were sufficient to produce significant stimulation of cartilage matrix gene expression and subsequent proteoglycan production. Moreover, cells in infected cultures maintained a chondrocytic phenotype and continued to express elevated insulinlike growth factor-I levels during 28 days of monolayer culture. Minimal synthetic activity, other than insulinlike growth factor-I ligand synthesis, was evident in synovial cultures. These experiments suggest several avenues for insulinlike growth factor-I supplementation of articular cartilage, including preimplantation adenoviral-insulinlike growth factor gene transfer to chondrocytes or chondroprogenitor cells, and direct injection of adenoviral-insulinlike growth factor to transfect the synovial structures in situ.