Regional Gene Therapy Research Articles

Arterial port catheter system placement using a translumbar aortic route.

Implantable ports for arterial infusion hold promise in a growing number of clinical applications, including regional chemotherapy and gene therapy. Percutaneous placement of arterial infusion ports has been described clinically; however, existing sites of access pose unique anatomic challenges. The feasibility of translumbar aortic placement of a port-catheter system for long-term arterial infusion was assessed. Eight swine underwent placement of a percutaneously implantable arterial port-catheter system using a translumbar aortic approach for catheter placement. The tip of the catheter was positioned in the left or right hepatic artery and the port reservoir implanted in the paraspinal subcutaneous tissues. Patency and position of the port-catheter system were assessed at 7 to 10 days and 28 days. The insertion procedure was modified in two animals by introducing tissue adhesive around the catheter to prevent dislodgment. Immediate technical success was achieved in 6 of 8 animals (75%). Catheters were dislodged into the aorta in two animals on the day of the procedure, extravascular in four animals and intra-aortic in one animal by day 10. One animal died at 21 days of sepsis. The port-catheter systems remained functional at 28 days with catheters positioned within the left or right hepatic artery in three animals, including two animals in which the catheter had been fixated within the paraspinal soft tissues with tissue adhesive. A translumbar aortic route for percutaneous placement of arterial infusion port-catheter systems is feasible in swine and holds potential as an ancillary access site in a clinical setting if the obstacle of catheter fixation can be overcome.

Hepatic intra-arterial delivery of a retroviral vector expressing the cytosine deaminase gene, controlled by the CEA promoter and intraperitoneal treatment with 5-fluorocytosine suppresses growth of colorectal liver metastases.

Targeting of colorectal liver metastases by regional gene therapy was tested in a clinically relevant syngeneic model. First, the CEA-CD-113 retroviral vector containing the cytosine deaminase gene controlled by the CEA specific tumour cell promoter, was shown in vitro to convert 5-fluorocytosine to 5-fluorouracil, resulting in cancer cell killing with a large bystander effect. Second, 10 days after the establishment of liver metastases, retroviral vectors were delivered to the liver by hepatic artery injection. After 5-fluorocytosine administration for 7 days, most surface metastases disappeared and tumour volumes were suppressed up to 8.2-fold. The results support the development of this approach for patient treatment.

Poly(lactide-co-glycolide)/hydroxyapatite delivery of BMP-2-producing cells: a regional gene therapy approach to bone regeneration

Currently, functional treatment of fracture non-unions and bone loss remains a significant challenge in the field of orthopaedic surgery. Tissue engineering of bone has emerged as a new treatment alternative in bone repair and regeneration. Our approach is to combine a polymeric matrix with a cellular vehicle for delivery of bone morphogenetic protein-2 (BMP-2), constructed through retroviral gene transfer. The objective of this study is to develop an osteoinductive, tissue-engineered bone replacement system by culturing BMP-2-producing cells on an osteoconductive, biodegradable, polymeric-ceramic matrix. The hypothesis is that retroviral gene transfer can be used effectively in combination with a biodegradable matrix to promote bone formation. First, we examined the in vitro attachment and growth of transfected BMP-producing cells on a PLAGA–HA scaffold. Second, the bioactivity of the produced BMP in vitro was evaluated using a mouse model. It was found that the polymer-ceramic scaffold supported BMP-2 production, allowing the attachment and growth of retroviral transfected, BMP-2-producing cells. In vivo, the scaffold successfully functioned as a delivery vehicle for bioactive BMP-2, as it induced heterotopic bone formation in a SCID mouse model.

Biologic alternatives to stents and grafts.

The quest for biologic alternatives to stents and arterial grafts is a source of intense research at the basic and preclinical level. Several new devices and conduits currently under investigation may extend the uses of both stents and grafts beyond their current revascularization role into newer applications in the fields of regional pharmacology and gene therapy. This exciting prospect, although still unrealized, has general medical implications beyond cardiovascular disease. Development of such synergies between device, grafts, pharmacologic, molecular, and tissue engineering research is essential if the burgeoning data on new therapeutic genes is to be harnessed for clinical benefit.

Osteoinductive applications of regional gene therapy: ex vivo gene transfer.

Gene therapy represents the new frontier of medical science. Currently, there are no completely satisfactory treatment options for bone repair problems such as fracture nonunion, revision total joint arthroplasty, tumor resections, and fusions of the spine. Autogenous bone grafts, allograft implants, and prosthetic implants have been used to treat these problems. However, there are significant limitations associated with these methods including limited supply and limited osteogenic potential. Gene therapy, involving the manipulation of endogenous cells to generate specific proteins, offers a potential solution for these problems. By transferring genes into cells at a specific anatomic site, the osteoinductive properties of growth factors can be used at physiologic doses for a sustained period to facilitate a more significant healing response. Successful gene therapy involves four key steps: transduction, transcription, translation, and expression. To achieve gene transduction of a target cell, gene therapy models use vectors to enhance the entry and expression of exogenous deoxyribonucleic acid into the target cell's nucleus. The transduction of a gene can be performed via either an ex vivo or an in vivo approach. Although there are many potential target cells for gene therapy, the specific anatomic site, the quality of the bone, and the soft-tissue envelope, will influence the selection of the target cells for regional gene therapy. Gene therapy vectors delivered to a treatment site in osteoconductive carriers have yielded promising results. Several investigators have shown exciting results using ex vivo and in vivo regional gene therapy in animal models. Comparative studies and human clinical trials have not yet been performed but are necessary to identify the optimal genes and dosages for each specific application of regional gene therapy. In the future, the treatment options for bone loss problems will represent a clinical continuum based on the anatomic site, the condition of the target tissue bed, and the desired duration of protein production.

Current Management of Colorectal Liver Metastases

Colorectal cancer metastatic to the liver represents an uncommon situation in surgical oncology in which metastasectomy can effect longevity and may lead to cure. Liver resection can be done safely and has drastically improved 5-year survival rates to upwards of 35%, but only a minority of patients is eligible. Advances in imaging techniques facilitate detection of metastases and improve patient selection. For unresectable patients, and as an adjunct to resection, local disease control may be achieved with cryosurgery, radiofrequency ablation, or regional chemotherapy delivered by way of infusion through the hepatic artery. Areas of ongoing investigation include regional gene therapy and interference with tumor growth by inhibition of angiogenesis.

Antitumoral effect of interleukin-12-secreting fibroblasts in a mouse model of ovarian cancer: implications for the use of ovarian cancer biopsy-derived fibroblasts as a vehicle for regional gene therapy.

Fibroblasts can easily be cultured from human solid ovarian tumor biopsies and are readily transfected using retroviral vectors. To test the feasibility of using these cells as a vehicle for regional, intraperitoneal (i.p.) ovarian cancer gene therapy, we first examined the behavior of murine fibroblasts transduced with the neoR marker gene and injected i.p. in syngeneic mice. We found that these fibroblasts were not invasive and formed clusters preferentially attached to the pancreatic and hepatic mesentery, where they survived for at least 30 days and continued to express neoR. We subsequently assessed the effect of regional delivery of murine interleukin-12 (mIL-12)-secreting fibroblasts in a syngeneic immunocompetent mouse model of ovarian cancer (ID8). ID8 ovarian tumor cells were injected i.p. into one flank. Our results show a survival of 88% at 120 days postinjection of animals treated with mIL-12-secreting fibroblasts i.p. in the other flank on the same day as injection of tumor cells. At that time, all animals coinoculated with ID8 cells and unmodified fibroblasts had been or needed to be culled because of advanced neoplastic disease (P < 10(-5), log-rank test). Furthermore, animals treated with mIL-12-secreting fibroblasts had a statistically significant decrease in tumor burden, as measured by diaphragm weight, relative to mock-treated groups (P = .0032, H-test). Our histological data show evidence for both immunological and anti-angiogenic mechanisms being implicated in this antitumoral effect. In addition, no signs of IL-12-induced toxicity were observed in any of the tissue sections analyzed. Taken together, our findings suggest that ovarian tumor biopsy-derived fibroblasts are a safe and efficacious vehicle for i.p. delivery of IL-12 as a regional secondary treatment for minimal residual disease of ovarian cancer.

Fibroblast growth factor 2-retargeted adenoviral vectors exhibit a modified biolocalization pattern and display reduced toxicity relative to native adenoviral vectors.

Targeted vectors provide a number of advantages for systemic and local gene delivery strategies. Several groups have investigated the utility of using various ligands to alter the tropism of adenovirus (Ad) vectors. We have previously demonstrated that fibroblast growth factor (FGF) ligands can specifically target DNA transfection and Ad transduction through high-affinity FGF receptors (FGFRs). FGFRs are overexpressed in abnormally proliferating tissues, such as malignancies. The present studies explore the effects of retargeting with FGF2 on the tissue localization pattern and the systemic toxicity of Ad in mice. Results of semiquantitative PCR analyses indicate that the distribution of FGF2-Ad vector genome sequences after intravenous administration in mice is altered. Markedly lower amounts (10- to 20-fold) of FGF2-Ad localize to the liver when compared with native Ad. This decrease in liver deposition translates into a significant reduction in subsequent toxicity as measured by serum transaminases and histopathology in mice injected with FGF2-AdHSV-thymidine kinase with and without ganciclovir administration. In an intraperitoneal model of ovarian cancer, FGF2-Ad generates increased transgene expression in tumor tissue when compared with Ad. Taken together, these results indicate that the retargeting of Ad with FGF2 results in a more efficient vector system for systemic and regional gene therapy applications, with concomitant lower levels of systemic toxicity.

A phase I/II study of hepatic artery infusion with wtp53-CMV-Ad in metastatic malignant liver tumours.

Colorectal cancer (CRC) is the second commonest cause of cancer death in the UK, with greater than 40% of these patients destined to die of the disease despite current medical management. Death is commonly due to liver metastases with sequelae including progressive liver dysfunction. Most patients with liver metastases present with tumours that are unresectable and incurable with existing therapies. The median survival for CRC patients after diagnosis with liver metastases is approximately 6 months or less. The human p53 gene is a tumour suppressor gene involved in the control of cell proliferation. Loss of wild-type p53 function is associated with the uncontrolled growth of many types of human cancers. The reintroduction and expression of wild-type p53 into p53 altered tumour cells has been shown to suppress tumour growth or induce apoptosis in both in vitro and in vivo models. In our experience greater than 50% of CRC tumours have p53 alterations. This study seeks to evaluate the safety, biological efficacy and the effectiveness of wtp53-CMV-Ad treatment which is a recombinant adenoviral vector containing the wild-type human p53 gene. It will be administered by infusion via the hepatic artery, for the regional gene therapy of malignant liver tumours. Study patients will have incurable metastatic (CRC) malignant tumours of the liver with evidence of p53 alteration in their liver tumours. In vitro studies have demonstrated p53-specific antiproliferative effects of wtp53-CMV-Ad on human liver tumour cells and in vivo studies have demonstrated p53-specific antiproliferative effects on human liver tumour cells. The vector Ad-p53 is a recombinant, replication-defective adenovirus based on adenovirus serotype 5. It contains a sequence encoding wild-type p53 whose expression is under the control of the human cytomegalovirus immediate early promoter-enhancer. This construct will be growth in 293 cells which contain the adenoviral E1A and E1B coding sequences which have been removed from the vector to render it replication defective. The study design is an open-label, non-randomised, single-dose, dose escalation Phase I/II clinical trial anticipated to involve a maximum of 19 patients. wtp53-CMV-Ad will be administered by infusion in a reservoir connected to the hepatic artery, for regional gene therapy (surgically implanted pump) in 3 escalating doses to successive cohorts of 3 patients each until the maximum tolerated dose is determined. Subsequently, 10 patients will be treated with this dose. Regional wtp53-CMV-Ad therapy will be administered as a single bolus infusion via hepatic artery catheter. The route of administration of wtp53-CMV-Ad via hepatic artery infusion is designed to maximise gene therapy exposure to the malignant tumours while minimising exposure to normal tissues outside the liver. The clinical protocol is designed to monitor treatment toxicity. Another objective is to evaluate the biological efficacy, including efficiency and stability of gene transfer by analysis of tumour tissues following therapy. As an important part of this objective the pharmacokinetics of wtp53-CMV-Ad will be studied. Clinical evidence of anti-tumour efficacy will also be collected. In addition, the safety and efficacy of different doses levels of wtp53-CMV-Ad will be studied.

Stabilized plasmid-lipid particles for regional gene therapy: formulation and transfection properties.

Previous work (Wheeler et al, Gene Therapy 1999; 6: 271-281) has shown that plasmid DNA can be entrapped in 'stabilized plasmid-lipid particles' (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating. The PEG moieties are attached to a ceramide anchor containing an arachidoyl acyl group (PEG-CerC20). These SPLP exhibit low transfection potencies in vitro, due in part to the long residence time of the PEG-CerC20 on the SPLP surface. In this work we employed SPLP stabilized by PEG attached to ceramide containing an octanoyl acyl group (PEG-CerC8), which is able to quickly exchange out of the SPLP, to develop systems that give rise to optimized in vitro and in vivo (regional) transfection. A particular objective was to achieve cationic lipid contents that give rise to maximum transfection levels. It is shown that by performing the dialysis procedure in the presence of increasing concentrations of citrate, SPLP containing up to 30 mol% of the cationic lipid dioleoydimethylammonium chloride (DODAC) could be generated. The SPLP produced could be isolated from empty vesicles by sucrose density gradient centrifugation, and exhibited a narrow size distribution (62 +/- 8 nm, as determined by freeze-fracture electron microscopy) and a high plasmid-to-lipid ratio of 65 microg/micromol (corresponding to one plasmid per particle) regardless of the DODAC content. It was found that isolated SPLP containing 20-24 mol% DODAC resulted in optimum transfection of COS-7 and HepG2 cells in vitro, with luciferase expression levels comparable to those achieved for plasmid DNA-cationic lipid complexes. In vivo studies employing an intraperitoneal B16 tumor model and intraperitoneal administration of SPLP also demonstrated maximum luciferase expression for DODAC contents of 20-24 mol% and significantly improved gene expression in tumor tissue as compared with complexes. We conclude that SPLP stabilized by PEG-CerC8 and containing 20-24 mol% cationic lipid are attractive alternatives to plasmid DNA-cationic lipid complexes for regional gene therapy applications.

The effect of regional gene therapy with bone morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats.

Recombinant human bone morphogenetic proteins (rhBMPs) can induce bone formation, but the inability to identify an ideal delivery system limits their clinical application. We used ex vivo adenoviral gene transfer to create BMP-2-producing bone-marrow cells, which allow delivery of the BMP-2 to a specific anatomical site. The autologous BMP-2-producing bone-marrow cells then were used to heal a critical-sized femoral segmental defect in syngeneic rats. Femoral defects in five groups of rats were filled with 5 x 10(6) BMP-2-producing bone-marrow cells, created through adenoviral gene transfer (twenty-four femora, Group I); twenty micrograms of rhBMP-2 (sixteen femora, Group II); 5 x 10(6) beta-galactosidase-producing rat-bone-marrow cells, created through adenoviral gene transfer of the lacZ gene (twelve femora, Group III); 5 x 10(6) uninfected rat-bone-marrow cells (ten femora, Group IV); or guanidine hydrochloride-extracted demineralized bone matrix only (ten femora, Group V). Guanidine hydrochloride-extracted demineralized bone matrix served as a substrate in all experimental groups. Specimens that were removed two months postoperatively underwent histological and histomorphometric analysis as well as biomechanical testing. Twenty-two of the twenty-four defects in Group I (BMP-2-producing bone-marrow cells) and all sixteen defects in Group II (rhBMP-2) had healed radiographically at two months postoperatively compared with only one of the thirty-two defects in the three control groups (beta-galactosidase-producing rat-bone-marrow cells, uninfected rat-bone-marrow cells, and guanidine hydrochloride-extracted demineralized bone matrix alone). Histological analysis of the specimens revealed that defects that had received BMP-2-producing bone-marrow cells (Group I) were filled with coarse trabecular bone at two months postoperatively, whereas in those that had received rhBMP-2 (Group II) the bone was thin and lace-like. Defects that had been treated with bone-marrow cells producing beta-galactosidase (Group III), uninfected bone-marrow cells (Group IV), or guanidine hydrochloride-extracted demineralized bone matrix only (Group V) demonstrated little or no bone formation. Histomorphometric analysis revealed a significantly greater total area of bone formation in the defects treated with the BMP-2-producing bone-marrow cells than in those treated with the rhBMP-2 (p = 0.036). Biomechanical testing demonstrated no significant differences, with the numbers available, between the healed femora that had received BMP-2-producing bone-marrow cells and the untreated (control) femora with respect to ultimate torque to failure or energy to failure. This study demonstrated that BMP-2-producing bone-marrow cells created by means of adenoviral gene transfer produce sufficient protein to heal a segmental femoral defect. We also established the feasibility of ex vivo gene transfer with the use of biologically acute autologous short-term cultures of bone-marrow cells.

Regional gene therapy with a BMP-2-producing murine stromal cell line induces heterotopic and orthotopic bone formation in rodents.

The ability to continuously deliver osteoinductive proteins to a specific anatomic site would facilitate the treatment of fracture nonunions and other clinical problems associated with bone loss. We have developed a murine model of regional gene therapy. A bone-marrow stromal cell line infected with an adenovirus expressing recombinant bone morphogenetic protein-2 cDNA secreted biologically active bone morphogenetic protein-2. These bone morphogenetic protein-2-producing cells were able to induce abundant heterotopic bone formation when implanted into the quadriceps muscle of severe combined immune deficient mice and also successfully healed large segmental femoral defects in nude rats. These studies demonstrate that regional gene therapy with continuous delivery of osteoinductive factors to a specific anatomic site can enhance the formation and repair of bone.

Autologous fibroblasts as potential vehicle for regional ovarian cancer gene therapy.

The local delivery of cytokines to the site of a tumour inoculum has been reported to result in development of host anti-tumour immunity [1,2]. The majority of these approaches have used gene-modified tumour cells that act in a paracrine fashion to release cytokine in the tumour microenvironment. The enhanced immunogenicity displayed by these cytokine-secreting tumour cells, when used as vaccine, has resulted in the successful treatment of established tumours in selected models [3–5]. The use of autologous human tumour cells transduced to secrete different cytokines is currently being investigated in clinical trials [6]. This strategy, however, has many obstacles precluding successful clinical application. Tumour cells may be difficult to culture and transfect, and selection of transfected cells may require prolonged culture and alter expression of nominal tumour antigens.