Targeted Gene Therapy Approaches Research Articles

Targeted gene therapy for rare genetic kidney diseases

Chronic kidney disease (CKD) is one of the leading causes of mortality worldwide because of kidney failure and the associated challenges of its treatment including dialysis and kidney transplantation. About one-third of CKD cases are linked to inherited monogenic factors, making them suitable for potential gene therapy interventions. However, the intricate anatomical structure of the kidney poses a challenge, limiting the effectiveness of targeted gene delivery to the renal system. In this review, we explore the progress made in the field of targeted gene therapy approaches and their implications for rare genetic kidney disorders, examining preclinical studies and prospects for clinical application. In vivo gene therapy is most commonly used for kidney-targeted gene delivery and involves administering viral and non-viral vectors through various routes such as systemic, renal vein and renal arterial injections. Small nucleic acids have also been used in preclinical and clinical studies for treating certain kidney disorders. Unexpectedly, hematopoietic stem and progenitor cells have been used as an ex vivo gene therapy vehicle for kidney gene delivery, highlighting their ability to differentiate into macrophages within the kidney, forming tunneling nanotubes that can deliver genetic material and organelles to adjacent kidney cells, even across the basement membrane to target the proximal tubular cells. As gene therapy technologies continue to advance and our understanding of kidney biology deepens, there is hope for patients with genetic kidney disorders to eventually avoid kidney transplantation.

Gene Therapy for Malignant and Benign Gynaecological Disorders: A Systematic Review of an Emerging Success Story.

Simple SummaryThis review discusses all the major advances in gene therapy of gynaecological disorders, highlighting the novel and potentially therapeutic perspectives associated with such an approach. It specifically focuses on the gene therapy strategies against major gynaecological malignant disorders, such as ovarian, cervical, and endometrial cancer, as well as benign disorders, such as uterine leiomyomas, endometriosis, placental, and embryo implantation disorders. The above therapeutic strategies, which employ both viral and non-viral systems for mutation compensation, suicide gene therapy, oncolytic virotherapy, antiangiogenesis and immunopotentiation approaches, have yielded promising results over the last decade, setting the grounds for successful clinical trials.Despite the major advances in screening and therapeutic approaches, gynaecological malignancies still present as a leading cause of death among women of reproductive age. Cervical cancer, although largely preventable through vaccination and regular screening, remains the fourth most common and most lethal cancer type in women, while the available treatment schemes still pose a fertility threat. Ovarian cancer is associated with high morbidity rates, primarily due to lack of symptoms and high relapse rates following treatment, whereas endometrial cancer, although usually curable by surgery, it still represents a therapeutic problem. On the other hand, benign abnormalities, such as fibroids, endometriosis, placental, and embryo implantation disorders, although not life-threatening, significantly affect women’s life and fertility and have high socio-economic impacts. In the last decade, targeted gene therapy approaches toward both malignant and benign gynaecological abnormalities have led to promising results, setting the ground for successful clinical trials. The above therapeutic strategies employ both viral and non-viral systems for mutation compensation, suicide gene therapy, oncolytic virotherapy, antiangiogenesis and immunopotentiation. This review discusses all the major advances in gene therapy of gynaecological disorders and highlights the novel and potentially therapeutic perspectives associated with such an approach.

CRISPR-targeted MAGT1 insertion restores XMEN patient hematopoietic stem cells and lymphocytes

AbstractXMEN disease, defined as “X-linked MAGT1 deficiency with increased susceptibility to Epstein-Barr virus infection and N-linked glycosylation defect,” is a recently described primary immunodeficiency marked by defective T cells and natural killer (NK) cells. Unfortunately, a potentially curative hematopoietic stem cell transplantation is associated with high mortality rates. We sought to develop an ex vivo targeted gene therapy approach for patients with XMEN using a CRISPR/Cas9 adeno-associated vector (AAV) to insert a therapeutic MAGT1 gene at the constitutive locus under the regulation of the endogenous promoter. Clinical translation of CRISPR/Cas9 AAV-targeted gene editing (GE) is hampered by low engraftable gene-edited hematopoietic stem and progenitor cells (HSPCs). Here, we optimized GE conditions by transient enhancement of homology-directed repair while suppressing AAV-associated DNA damage response to achieve highly efficient (>60%) genetic correction in engrafting XMEN HSPCs in transplanted mice. Restored MAGT1 glycosylation function in human NK and CD8+ T cells restored NK group 2 member D (NKG2D) expression and function in XMEN lymphocytes for potential treatment of infections, and it corrected HSPCs for long-term gene therapy, thus offering 2 efficient therapeutic options for XMEN poised for clinical translation.

Tau hyperphosphorylation in the retinal ganglion cells is attenuated upon silencing of SHP2 phosphatase

AbstractBackgroundHuman and animal studies demonstrate that both amyloid precursor protein and phosphorylated Tau which are hallmark proteins associated with Alzheimer’s disease (AD) pathology are perturbed in the retinal tissues. The accumulation of pTau in the retina in addition to the brain tissue suggests eye as a valuable structure for disease diagnosis and to study the disease mechanisms in neurodegenerative disease. Recent studies in human tissues and animal models have established a molecular overlap of AD with glaucoma in the eyes and both the diseases demonstrate altered amyloid beta and Tau phosphorylation profile. We show that Shp2 interacts with Tau protein in retina and silencing this phosphatase in the retinal ganglion cells using AAV2 mediated gene therapy can protect the retina.MethodAnimal model of ocular hypertension was generated by repeat administration of polystyrene beads into the anterior chamber of rat eyes for 8 weeks resulting in sustained elevation of intraocular pressure (20.84±1.65 mmHg). Tau expression changes were investigated both in the animal model and human tissues obtained from the Sydney Eye bank (n=3) using immunohistochemical and WB. Silencing of Shp2 expression in the inner retina was achieved using intravitreal administration of AAV2 constructs under the control of CAG hybrid promoter. The animals were monitored for 2 months after Shp2 silencing and effects on Tau protein expression studied (n=48).ResultIncreased Tau Ser404 phosphorylation was observed in both human glaucoma retinas (p<0.02; student t‐test) and in the animal model of glaucoma (p<0.05) in the WB and immunohistochemical analysis. Shp2 silencing (82±4%) was achieved using AAV2 administration and transduction was monitored using GFP staining. A 7.5±3‐fold downregulation Shp2 expression was achieved using shRNAmir that resulted in significant reduction of pTau Ser404 levels in the inner retina (p<0.028, Bornferroni post‐hoc test) indicating a regulatory effect of the phosphatase on the pTau levels.ConclusionThis study demonstrates that TauSer404 phosphorylation in the retina in vivo is regulated by Shp2 phosphatase. Targeted gene therapy approaches to modulate Shp2 expression can be used as a novel strategy to reduce Tau pathology in the neural tissues in various neurodegenerative disorders.

Targeted AAVP-based therapy in a mouse model of human glioblastoma: a comparison of cytotoxic versus suicide gene delivery strategies

Glioblastoma persists as a uniformly deadly diagnosis for patients and effective therapeutic options are gravely needed. Recently, targeted gene therapy approaches are reemerging as attractive experimental clinical agents. Our ligand-directed hybrid virus of adeno-associated virus and phage (AAVP) is a targeted gene delivery vector that has been used in several formulations displaying targeting ligand peptides to deliver clinically applicable transgenes. Here we compared different constructs side-by-side in a tumor model, an orthotopic model of xenograft human glioblastoma cells stereotactically implanted in immunodeficient mice. We have used divergent therapeutic strategies for two AAVP constructs, both displaying a double-cyclic RGD4C motif ligand specific for alpha V integrins expressed in tumor vascular endothelium, but carrying different genes of interest for the treatment of intracranial xenografted tumors. One construct delivered tumor necrosis factor (TNF), a purely cytotoxic gene for antitumor activity (RGD4C-AAVP-TNF); in the other construct, we delivered Herpes simplex virus thymidine kinase (HSVtk) for in tandem molecular-genetic imaging and targeted therapy (RGD4C-AAVP-HSVtk) utilizing ganciclovir (GCV) for a suicide gene therapy. Both AAVP constructs demonstrated antitumor activity, with damage to the tumor-associated neovasculature and induction of cell death evident after treatment. In addition, the ability to monitor transgene expression with a radiolabeled HSVtk substrate pre and post GCV treatment demonstrated the theranostic potential of RGD4C-AAVP-HSVtk. We conclude that targeted AAVP constructs delivering either cytotoxic TNF or theranostic HSVtk followed by suicide gene therapy with GCV have comparable preclinical efficacy, at least in this standard experimental model. The results presented here provide a blueprint for future studies of targeted gene delivery against human glioblastomas and other brain tumors.

557. Targeted CYBB Minigene Insertion into the CYBB Locus for Correction of X-CGD iPSCs Requires Intronic Elements for Expression

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency characterized by defective phagocyte production of microbicidal reactive oxygen species (ROS), resulting in recurring, life-threatening infections and hyper-inflammation. Mutations causing X-CGD span the entire 13 exons or intronic splice sites of the >30-kb CYBB gene encoding gp91phox, resulting in a loss of gp91phox protein expression. We previously tested a TALEN-mediated targeted gene therapy approach to insert a codon-optimized CYBB minigene into the start site of endogenous CYBB. Although targeted insertion into the endogenous start site was achieved in X-CGD patient iPSCs, little or no gp91phox expression or ROS activity was observed upon granulocyte differentiation, suggesting that downstream intronic or regulatory elements may be necessary for efficient gene expression from the CYBB promoter. To test this hypothesis, we tested CRISPR-mediated targeted insertion of a codon-optimized CYBB cDNA consisting of exons 2 through 13 (CYBB2-13) together with a puromycin-resistance gene cassette into exon 2 of the CYBB locus. In iPSCs from X-CGD patients with a CYBB mutation in exon 5, exon 7, or intron 10, the efficiency of targeted insertion of the CYBB2-13 plasmid donor without random inserts in puromycin-selected clones was 50-66%. Upon granulocyte differentiation of CYBB2-13 corrected X-CGD iPSCs, gp91phox expression and ROS production were restored to levels 64-100% (gp91phox) and 68-76% (DHR) of normal healthy donor controls. As expected for expression from the endogenous CYBB promoter, expression of gp91phox was specific to CD13+ granulocytes, and was undetected in undifferentiated iPSCs. This targeted gene therapy approach should allow correction of ~90% of X-CGD patient mutations (those involving mutations in exons 2 through 13), to restore ROS activity while maintaining normal regulation of CYBB expression. Further, these findings demonstrate a key issue for the design of targeted gene insertion to capture expression from an endogenous promoter: for some endogenous promoters, the inclusion of intronic elements is necessary for efficient expression of the insert.

Abstract 117: Development of a Hybrid Cryogel-coated Prosthetic Vascular Graft for Delivery of Targeted Gene Therapies

Objectives: Anastomotic neo-intimal hyperplasia (ANIH) remains a limiting factor in the long-term success of prosthetic vascular grafts. Understanding of the molecular mechanisms involved in the host response to prosthetic material has set the stage for using this material to deliver siRNA and other modulators of the ANIH-associated transcriptome response, along with antithrombotic agents. To create practical and effective drug delivery from a prosthetic material, we have combined a platform of polyethylene terephthalate (PET) fabric with an applied cryogel carrying the biologic agents, resulting in a bioactive prosthetic graft system (BPGS). Methods: Hybrid grafts were synthesized by cryopolymerization of methacrylated alginate and heparin onto electrospun, knitted, or woven PET. Arg-Gly-Asp peptides were added to increase cell adhesion. Scanning electron microscopy (SEM) was used to study the microstructure at 1 day, and 2, 4, and 8 weeks. Human aortic endothelial cell (HAoEC) adherence and proliferation were assessed with a resazurin-based assay, and confocal microscopy was used to visualize cell interaction with the graft material. Heparin activity of the material in buffer solution was measured using an anti-Xa assay. Results: SEM demonstrated large interconnected pores throughout the entire structure for all graft types, with minimal degradation of the cryogel after 8 weeks. HAoEC incorporated, adhered, and proliferated over 7 days for all materials (Figure 1). Anti-Xa assay confirmed continued activity of heparin from all grafts over 7 days. Conclusion: We have created a BPGS with a biocompatible cryogel polymer coating that allows for cell adherence and growth, with antithrombotic activity. This system allows application of the gel and biologic agents to PET prior to implantation, and provides flexibility in combining bioengineering and targeted gene therapy approaches to create an improved prosthetic graft adaptable to evolving strategies.

In vitro rescue of FGA deletion by lentiviral transduction of an afibrinogenemic patient's hepatocytes

Congenital afibrinogenemia is a rare inherited autosomal recessive disorder in which a mutation in one of three genes coding for the fibrinogen polypeptide chains Aα, Bβ and γ results in the absence of a functional coagulation protein. A patient with congenital afibrinogenemia, resulting from an FGA homozygous gene deletion, underwent an orthotopic liver transplant that resulted in complete restoration of normal hemostasis. The patient's explanted liver provided a unique opportunity to further investigate a potential novel treatment modality. To explore a targeted gene therapy approach for patients with congenital afibrinogenemia. At the time of transplant, the patient's FGA-deficient hepatocytes were isolated and transduced with lentiviral vectors encoding the human fibrinogen Aα-chain. FGA-transduced hepatocytes produced fully functional fibrinogen in vitro. Orthotopic liver transplantation is a possible rescue treatment for failure of on-demand fibrinogen replacement therapy. In addition, we provide evidence that hepatocytes homozygous for a large FGA deletion can be genetically modified to restore Aα-chain protein expression and secrete a functional fibrinogen hexamer.

Abstract 3321: Novel therapeutic approaches targeting the transferrin receptor 1 for the treatment of mantle cell lymphoma.

Abstract Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with no defined curative therapy. A hallmark of MCL is the high level of cyclin D1 expression that promotes cell cycle progression with the subsequent increase in proliferation, need for iron, and, thus, overexpression of the transferrin receptor 1 (TfR1). We previously developed an antibody-avidin fusion protein (ch128.1Av) targeting this receptor that shows an enhanced intrinsic in vitro cytotoxicity in malignant hematopoietic cells when compared to the parental antibody without avidin (ch128.1). This cytotoxicity is due to the ability of ch128.1Av to decrease cell surface TfR1 leading to lethal iron deprivation. However, both ch128.1Av and ch128.1 have shown anti-tumor activity in human xenograft models of multiple myeloma. ch128.1Av is also a universal delivery system of biotinylated agents. The main goal of this study is to evaluate the potential use of our anti-TfR1 antibodies alone and as delivery vehicles as possible MCL therapies. Screening of a panel of six human MCL cell lines (Mino, JVM-2, JVM-13, JeKo-1, Z-138, and REC-1) showed high expression of TfR1 but not TfR2, confirming specificity of TfR1 targeted therapy. We found that, when used alone, ch128.1Av exhibits cytotoxicity in MCL cells. In addition, the cytotoxicity dramatically increases when this fusion protein is conjugated to biotinylated saporin, a plant protein synthesis inhibitor. However, a concern with the systemic delivery of potent toxins is their potential side effects. To further enhance the delivery specificity, we explored the potential of dual targeted gene therapy approaches. The first level of targeting occurs through the use of our anti-TfR1 antibodies and takes advantage of the overexpression of the TfR1 on cancer cells. The second level of targeting occurs through transcriptional regulation using the immunoglobulin (Ig) promoter to restrict the expression of the delivered gene to malignant B-cells. Two different approaches were examined: 1) the use of the parental antibody (ch128.1) complexed to lentivirus with a modified, pseudotyped Sindbis virus envelope protein containing the ZZ domain of protein A; and 2) the use of ch128.1Av conjugated to a similar virus containing the biotin acceptor peptide (instead of the ZZ domain) that allows for biotinylation of the virus. We found that the reporter gene encoding enhanced green fluorescent protein (EGFP), under the control of the Ig promoter, was expressed in human malignant B cells U266 (multiple myeloma) and Z-138 (MCL), but not in Jurkat (human T-cell leukemia) cells. As expected, EGFP was expressed in all cell lines when a ubiquitous promoter was used. These results confirm the dual targeted nature of our approaches and suggest the potential use of our anti-TfR1 antibodies alone or as delivery vehicles as therapeutic interventions against incurable B-cell malignancies such as MCL and multiple myeloma. Citation Format: Lai Sum Leoh, Michael A. Erb, Kouki Morizono, Irvin S. Y. Chen, Manuel L. Penichet, Tracy R. Daniels-Wells. Novel therapeutic approaches targeting the transferrin receptor 1 for the treatment of mantle cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3321. doi:10.1158/1538-7445.AM2013-3321

Determining Limitations in Human CD34+ Cell Transduction with An HIV1-Based Lentiviral Vector,

Abstract 4171Hematopoietic stem cell (HSC)-targeted gene therapy approaches utilizing HIV1-based lentiviral vectors is a promising modality for a number of inherited and acquired disorders affecting the blood. Unlike cell lines and murine HSCs, human HSCs demonstrate lower transduction efficiency with HIV1 vectors, potentially limiting this approach. Lentiviral transduction requires four essential steps: (1) internalization, (2) reverse transcription, (3) nuclear transport, and (4) integration into genomic DNA. In this study, we sought to evaluate which step limits transduction of human CD34+ cells with HIV1 vectors.We transduced HeLa cells and human CD34+ cells with a self-inactivating-HIV1 vector at low and a 10-fold-higher multiplicity of infection (MOI) (MOI 0.5 vs. MOI 5 for HeLa cells and MOI 5 vs. MOI 50 in CD34+ cells, respectively) and assayed each of the four steps based upon the rationale that if a given step was not rate limiting, a 10-fold-greater value would be obtained at the 10-fold-higher MOI. Amounts of the vectors were determined by real time PCR using RNA and DNA samples extracted from the transduced cells over time. The ratios of vector genome amounts at high MOI to amounts at low MOI were compared between CD34+ cells and HeLa cells to determine whether there was a limiting step for CD34+ cell transduction.To evaluate RNA internalization, we determined relative amounts of vector RNA in the cells at 5 min, 10 min, 30 min, and 60 min after transduction. Among HeLa cells, relative amounts of vector RNA showed a rapid increase and then plateaued at 30 min after transduction, while in the CD34+ cells, relative amounts of vector RNA increased gradually over 60 min. When we calculated the ratios of vector RNA amounts at high MOI to low MOI for each type of cell, we observed similar ratios when comparing HeLa to CD34+ cells at 60 min after transduction. These data suggest that despite slower internalization of vector RNA in CD34+ cells, it is not a limiting step in lentiviral transduction.To evaluate reverse transcription, nuclear transport, and integration; we determined relative amounts of vector DNA at 2 hrs, 4 hrs, 6 hrs, 12 hrs, 24 hrs, 2 days, 3 days, 6 days, and 10 days after transduction. We detected similar patterns of relative vector DNA in both Hela and CD34+ cells: as their relative amounts increased, they reached a peak at 24 hrs (reverse transcription), decreased, and then reached a plateau at 2–3 days after transduction (nuclear transport and integration). CD34+ cells showed higher ratios of vector DNA at high MOI to low MOI at 2–24 hrs, similar ratios at 2 days, and lower ratios at 3–10 days, compared to HeLa cells (Figure). These data suggest that transduction of human CD34+ cells has a limitation around 2 days after vector exposure corresponding to nuclear transport or integration.To evaluate nuclear transport for both types of cells, we compared the relative amounts of vector DNA to total DNA and nuclear DNA. Nuclear DNA was extracted from the nuclear fractions of transduced cells which had been isolated by a sucrose gradient. In both cells, the nuclear DNA had tendencies to show lower amounts of vector DNA at 6–48 hrs after transduction and similar or higher amounts of vector DNA at 2–7 days, compared to the total DNA. These data suggest that vector DNA is transported to the nucleus in about 2–3 days.Taken together, these data suggest that nuclear transport is a limiting step in lentiviral transduction of human CD34+ cells. We hypothesized that transduction efficiency for human CD34+ cells might increase under conditions that induce greater cell expansion, since dividing cells do not have intact nuclear membranes. We compared relative amounts of vector DNA in human CD34+ cells transduced in X-VIVO10 medium (standard) and StemlineII medium (greater expansion) at 12 hrs, 24 hrs, 2 days, 3 days, 5 days, and 7 days after transduction. StemlineII exposed cells showed higher amounts of vector DNA, compared to X-VIVO10, at all time points except 24 hrs. The data suggest that StemlineII increases transduction efficiency for human CD34+ cells by increasing nuclear transport.In summary, we demonstrate that nuclear transport is a limiting step in lentiviral transduction of human CD34+ cells and that StemlineII medium could increase transduction efficiency. These data are helpful for the design of strategies to improve upon lentiviral transduction for human CD34+ cells by improving nuclear transport. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Efficacious and Safe Tissue-Selective Controlled Gene Therapy Approaches for the Cornea

Untargeted and uncontrolled gene delivery is a major cause of gene therapy failure. This study aimed to define efficient and safe tissue-selective targeted gene therapy approaches for delivering genes into keratocytes of the cornea in vivo using a normal or diseased rabbit model. New Zealand White rabbits, adeno-associated virus serotype 5 (AAV5), and a minimally invasive hair-dryer based vector-delivery technique were used. Fifty microliters of AAV5 titer (6.5×1012 vg/ml) expressing green fluorescent protein gene (GFP) was topically applied onto normal or diseased (fibrotic or neovascularized) rabbit corneas for 2-minutes with a custom vector-delivery technique. Corneal fibrosis and neovascularization in rabbit eyes were induced with photorefractive keratectomy using excimer laser and VEGF (630 ng) using micropocket assay, respectively. Slit-lamp biomicroscopy and immunocytochemistry were used to confirm fibrosis and neovascularization in rabbit corneas. The levels, location and duration of delivered-GFP gene expression in the rabbit stroma were measured with immunocytochemistry and/or western blotting. Slot-blot measured delivered-GFP gene copy number. Confocal microscopy performed in whole-mounts of cornea and thick corneal sections determined geometric and spatial localization of delivered-GFP in three-dimensional arrangement. AAV5 toxicity and safety were evaluated with clinical eye exam, stereomicroscopy, slit-lamp biomicroscopy, and H&E staining. A single 2-minute AAV5 topical application via custom delivery-technique efficiently and selectively transduced keratocytes in the anterior stroma of normal and diseased rabbit corneas as evident from immunocytochemistry and confocal microscopy. Transgene expression was first detected at day 3, peaked at day 7, and was maintained up to 16 weeks (longest tested time point). Clinical and slit-lamp eye examination in live rabbits and H&E staining did not reveal any significant changes between AAV5-treated and untreated control corneas. These findings suggest that defined gene therapy approaches are safe for delivering genes into keratocytes in vivo and has potential for treating corneal disorders in human patients.

Modulation of the effectors of radiation fibrosis: A targeted gene therapy approach

Modulation of the effectors of radiation fibrosis: A targeted gene therapy approach

Replication-deficient rSV40 mediate pancreatic gene transfer and long-term inhibition of tumor growth

Pancreatic cancer is one of the most aggressive and devastating human malignancies. There is an urgent need for more effective therapy for patients with advanced disease. In this context, genetic therapy potentially represents a rational new approach to treating pancreatic cancer, which could provide an adjunct to conventional options. Because of the promise of recombinant SV40 vectors, we tested their ability to deliver a transgene, and to target a transcript, so as to inhibit pancreatic tumors growth in vivo. BxPC3 and Capan-1 cells were efficiently transduced using SV40 vectors without selection, as compared to synthetic vectors PEI. SV40 vectors were as efficient as adenoviral vectors, and provided long-term transgene expression. Next, we devised a SV40-derived, targeted gene therapy approach of pancreatic cancer, by combining hTR tumor-specific promoter with sst2 somatostatin receptor tumor-suppressor gene. In vitro cell proliferation was strongly impaired following administration of SV(hTR-sst2). SV40-derived sst2-mediated antiproliferative effect was dependent on the local production of somatostatin. In vivo, intratumoral gene transfer of sst2 using rSV40 vectors resulted in a marked inhibition of Capan-1 tumor progression, and proliferation. These results represent the initial steps toward a novel approach to the gene therapy of pancreatic cancer using SV40 as a vector.

A multi-mutant herpes simplex virus vector has minimal cytotoxic effects on the distribution of filamentous actin, alpha-actinin 2 and a glutamate receptor in differentiated PC12 cells.

To develop effective gene therapy techniques that target populations of neurons in the spinal cord, suitable vectors must be developed that will undergo efficient, retrograde transport from an appropriate peripheral site and will not be cytotoxic. Our previous work (LeVatte et al, 1998a) has demonstrated that a replication defective herpes simplex virus vector 14Hdelta3vhsZ, that has been substantially detoxified, is retrogradely transported from peripheral sites and can infect large numbers of the targeted spinal neurons. We plan to develop targeted gene therapy approaches designed to modulate the excitatory glutamatergic methyl-D-aspartate (NMDA) receptor in spinal cord neurons as a means of ameliorating a form of episodic high blood pressure that occurs after spinal cord injury. In this report, we demonstrate that, in differentiated PC12 cells, a neuronal-like cell line, the virus vector does not appear to alter aspects of the cytoskeletal architecture important to the proper distribution of the NMDA receptor. In turn, the distribution of endogenous NMDA receptor 1 subunit protein (NMDAR1) or a transfected NMDAR1-green fluorescent fusion protein was also found to be unaltered after vector infection. However, whereas endogenous NMDAR1 distribution was maintained, vector infection did tend to reduce the level of its expression. This drop in endogenous NMDAR1 expression coincided with the expression of the HSV immediate early genes ICP0 and ICP27 over the first 24-48 h. These results indicate that the 14Hdelta3vhsZ herpes simplex virus vector is suitable to use in future strategies to alter the level of gene expression in targeted populations of spinal cord neurons.

Targeted gene therapy.

Melanin biosynthesis is limited to melanocytes partly as a consequence of transcriptional regulation of the enzymes involved in this pathway. Promoter sequences of these enzyme genes may be utilised to drive expression of complementary DNA coding for therapeutic genes so as to provide transcriptional targeting. We have used the 5'-flanking sequences of the murine tyrosinase or tyrosinase-related protein 1 (TRP-1) genes to show that such transcriptional targeting can be achieved both in vitro and in vivo. Using IL-2 as an example of an immunostimulatory gene and Herpes Simplex Virus thymidine kinase (HSVtk) as an example of a prodrug-activating gene we have shown, in murine model systems, that substantial anti-tumour effects can be achieved by targeted gene therapy approaches. The stage now is set for initial clinical evaluations in human patients.