Targeted Gene Transfer Research Articles

Recent progress on technologies and applications of transgenic poultry

Manipulation of the poultry genome has the potential to improve poultry production and offer a powerful bioreactor for the production of pharmaceutical and industrial proteins in eggs. However, before realizing this, the methods for producing transgenic poultry must become routine. The direct modification of an embryo with DNA or viral vectors is possible, but this approach does not have the ability to make locus-specific modifications to the genome. To overcome this problem, several cell-based protocols, which use mainly blastodermal cells (BDCs), embryonic stem cells (ESCs), primordial germ cells (PGCs) and spermatogonial stem cells (SSCs) have been developed to generate transgenic chickens. At present, the complete system for isolating, expanding, transfecting, selecting and re-expanding embryonic stem cell cultures and the subsequent production of high-grade somatic chimeras has been reported, but germline chimeras with transgenic progeny have not yet been achieved. Although the use of viral systems can achieve highly efficient gene transfer, the potential safety issues may limit their practical application. Among the many possible permutations and combinations of target cell and gene transfer methods described in this review, targeting SSCs in vitro using non-viral-based gene transfer and re-injecting them to cock testis is the most efficient and cost-effective strategy to produce transgenic poultry. Key words: Genetic modification, embryo, blastodermal cells, primordial germ cells, stem cells, viral vector.

Corpus Callosum: a Favorable Target for rSFV-Mediated Gene Transfer to Rat Brain with Broad and Efficient Expression

Recombinant Semliki Forest virus (rSFV), as a new kind of neurotropic vector system, has great potential of gene therapy for stroke. However, very little is known about its transduction characteristics in cerebral cortex or corpus callosum (CC) in vivo, which are common targets for gene transfer in experimental stroke therapy. Here, we investigate and compare rSFV-mediated gene expression at above two brain regions in rat; 2.0 x 10(7) IU of rSFV encoding green fluorescent protein (rSFV-GFP) was locally injected into CC or cerebral cortex in two groups. At 36 h following injection, the number of GFP-positive cells, GFP distribution volume, and GFP expression level were examined in the rat brain of each group using continuous frozen sections and enzyme-linked immunosorbent assay. rSFV vector displayed noticeably different transduction patterns in CC and cerebral cortex in vivo. CC injection of vector increased GFP-positive cell number by 802%, GFP transduction volume by 958%, and GFP expression level by 508% compared with cortical injection (all P < 0.01). We concluded that rSFV CC delivery significantly enhances transduction efficiency in rat brain with its ability to achieve transgene extensive transduction and abundant expression, and CC may be a favorable target for improving rSFV-based gene delivery efficiency to brain.

Salmonella -mediated delivery of RNase P-based ribozymes for inhibition of viral gene expression and replication in human cells

A fundamental challenge in gene therapy is to develop approaches for delivering nucleic acid-based gene interfering agents, such as small interfering RNAs and ribozymes, to the appropriate cells in a way that is tissue/cell specific, efficient, and safe. Using human cytomegalovirus (HCMV) infection of differentiated macrophages as the model, we showed that Salmonella can efficiently deliver RNase P-based ribozyme sequence in specific human cells, leading to substantial ribozyme expression and effective inhibition of viral infection. We constructed a functional RNase P ribozyme (M1GS RNA) that targets the overlapping mRNA region of two HCMV capsid proteins, the capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. Substantial expression of ribozymes was observed in human differentiated macrophages that were treated with attenuated Salmonella strains carrying the ribozyme sequence constructs. A reduction of 87-90% in viral CSP expression and a reduction of about 5,000-fold in viral growth were observed in cells that were treated with Salmonella carrying the sequence of the functional ribozyme but not with those carrying the sequence of a control ribozyme that contained mutations abolishing the catalytic activity. To our knowledge, this study showed for the first time that ribozymes expressed following targeted gene transfer with Salmonella-based vectors are highly active and specific in blocking viral infection. Moreover, these results demonstrate the feasibility to develop Salmonella-mediated gene transfer of RNase P ribozymes as an effective approach for gene-targeting applications.

Analysis of a primate cone mosaic following targeted gene transfer using recombinant adeno-associated virus vectors

Analysis of a primate cone mosaic following targeted gene transfer using recombinant adeno-associated virus vectors

Electrically Mediated Delivery of Plasmid DNA to the Skin, Using a Multielectrode Array

The easy accessibility of skin makes it an excellent target for gene transfer protocols. To take full advantage of skin as a target for gene transfer, it is important to establish an efficient and reproducible delivery system. Electroporation is a strong candidate to meet this delivery criterion. Electroporation of the skin is a simple, direct, in vivo method to deliver genes for therapy. Previously, delivery to the skin was performed by means of applicators with relatively large distances between electrodes, resulting in significant muscle stimulation and pain. These applicators also had limitations in controlling the directionality of the applied field. To resolve this issue, a system consisting of an array of electrodes that decreased the distance between them and that were independently addressable for directional control of the field was developed. This new multielectrode array (MEA) was compared with an established electrode. In a rat model, comparable reporter expression was seen after delivery with each electrode. Delivery was also evaluated in a guinea pig model to determine the potential of this approach in an animal model with skin thickness and structure similar to human skin. The results clearly showed that effective delivery was related to both the electrode and the parameters chosen. With the MEA, the muscle twitching associated with application of electric fields was notably reduced compared with conventional electrode systems. This is important, as it will facilitate the translation of electroporation-mediated gene delivery to skin for clinical use with DNA vaccines or for therapies for cancer or protein deficiencies.

Gene therapy for acute myeloid leukemia using Sindbis vectors expressing a fusogenic membrane glycoprotein

AML has a dismal prognosis. It was previously shown that the expression of gene coding for the hyperfusogenic gibbon ape leukemia virus envelope glycoprotein (GALV.fus) can efficiently kill leukemic cells. However, target killing effect of GALV.fus on leukemia cells may be limited. Viral vectors, such as retroviruses and adenoviruses, have been developed to deliver heterologous genes into tumors in vivo, but these vectors have some limitations for gene therapy of leukemia. Another virus that has drawn interest as a gene transfer vector is the Sindbis virus. Sindbis virus efficiently infects human tumor cells through the high-affinity 67 kDa Laminin receptor. We found that Laminin-R was obviously expressed in HL-60 and primary human AML cells，but weakly expressed in K562 cells and blood samples of normal human. So we reasoned that Sindbis-virus-based vectors might be ideal for target gene transfer of GALV.fus to acute myeloid leukemic cell. It was shown that Sindbis virus efficiently transduced human acute myeloid leukemic cells with high expression of Laminin-R and exhibited potent cytopathic potential. What is more, we found that CFU-GMs were significantly reduced after Sindbis virus carrying GALV.fus transduced human primary AML cells. Sindbis virus carrying GALV.fus was active against human AML xenografts in vivo. Taken together, we concluded that Sindbis virus carrying GALV.fus may be an useful strategy for gene therapy of acute myeloid leukemia.

Targeted integration and removal of transgenes in hybrid aspen (Populus tremula L. x P. tremuloides Michx.) using site‐specific recombination systems

Two site-specific recombination systems, Cre/lox and FLP/FRT, were tested for marker gene removal and targeted gene transfer in a model tree system. A hybrid aspen clone (Populus tremula x Populus tremuloides) was co-transformed with plasmids containing either the FLP or the Cre recombinase, both under control of a heat-inducible promoter (HSP, Gmhsp17.5-E from soybean) flanked by the two recognition sites (FRT or lox). Molecular investigations of heat-shock treated Cre or FLP transgenic lines indicate excision of inserts between the two recognition sites. Further, a site-specific recombination at the FRT sites leading to targeted integration of a fragment could be demonstrated for the FLP/FRT system. Transgenic aspen carrying two constructs (each with different genes between the FRT sites) revealed (i) excision of both fragments between the FRT sites, and (ii) targeted integration of the fragment from the second construct exactly at the former position of the fragment in the first construct. These results indicate the usefulness of the two site-specific recombination systems in the tree species Populus. Combining both site-specific recombination systems, a strategy is suggested for targeted transgene transfer and removal of antibiotic marker genes.

Targeted Gene Delivery to Selected Liver Segments Via Isolated Hepatic Perfusion

Development of targeted gene transfer technologies is essential for in vivo gene therapy. In this study, we examined the feasibility of physically targeting an adenoviral vector to selected liver segments in rats by isolating the hepatic perfusion (IHSP) and clamping the portal vein between the upper and lower segments. The rats were divided into two groups: IHSP group and the inferior vena cava (IVC) group. The adenoviral vector, which harbored the beta-galactosidase (beta-gal) gene, was administered via the portal vein, after which unbound vector particles were washed out with phosphate-buffered saline (PBS) and removed via the cannulated inferior vena cava (IVC) in IHSP group, while the IVC group received the transgene directly via the IVC without isolation of the hepatic perfusion. With this configuration (IHSP group), >99% of the beta-gal activity was limited to the targeted hepatic lobes, findings which were confirmed by histochemical staining with X-gal. We also found there to be significant differences in transgene expression among the hepatic lobes in the IVC group. Taken together, these results indicate that the IHSP technique is useful for local gene delivery to selected liver segments, and that when evaluating the efficacy of IHSP in the treatment of liver disease (e.g., nonresectable tumors), interlobar differences must be given careful consideration to ensure that sufficient drug or vector is delivered to all targeted hepatic lobes.

Targeted gene delivery into peripheral sensorial neurons mediated by self-assembled vectors composed of poly(ethylene imine) and tetanus toxin fragment c

A simple, safe and efficient system that can specifically transfect peripheral sensorial neurons can bring new answers to address peripheral neuropathies. A multi-component non-viral gene delivery vector targeted to peripheral nervous system cells was developed, using poly(ethylene imine) (PEI) as starting material. A binary DNA/polymer complex based on thiolated PEI (PEISH) was optimized, considering complex size and zeta potential and the ability to transfect a sensorial neuron cell line (ND7/23). The 50 kDa non-toxic fragment from tetanus toxin (HC), which has been previously shown to interact specifically with peripheral neurons and to undergo retrograde transport, was grafted to the complex core via a bifunctional PEG (HC-PEG) reactive for the thiol moieties present in the complex surface. Several formulations of HC-PEG ternary complexes were tested for targeting, by assessing the extent of cellular internalization and levels of transfection, in both the ND7/23 and NIH 3 T3 (fibroblast) cell lines. Targeted gene transfer to the neuronal cell line was observed for the complex formulations containing 5 and 7.5 μg of HC-PEG. Finally, our results demonstrate that the developed ternary vectors are able to transfect primary cultures of dorsal root ganglion dissociated neurons in a targeted manner and elicit the expression of a relevant neurotrophic factor.

Galactosyl conjugated N-succinyl-chitosan-graft-polyethylenimine for targeting gene transfer

Through incorporating lactobionic acid (LA) bearing a galactose group to N-succinyl-chitosan-graft-polyethylenimine (NSC-g-PEI), NSC-g-PEI-LA copolymers were synthesized as gene vectors with hepatocyte targeting properties. The molecular weight and composition of NSC-g-PEI-LA copolymers were characterized using gel permeation chromatography (GPC) and (1)H nuclear magnetic resonance spectroscopy ((1)H NMR) respectively. Agarose gel electrophoresis assays showed good DNA binding ability of NSC-g-PEI-LA, and the particle size of the NSC-g-PEI-LA/DNA complexes were between 150 and 400 nm as determined by a Zeta sizer. The NSC-g-PEI-LA/DNA complexes observed by scanning electron microscopy (SEM) exhibited a compact and spherical morphology. The zeta potentials of these complexes were increased with the weight ratio of NSC-g-PEI-LA/DNA. NSC-g-PEI-LA has a lower cytotoxicity than PEI (25 kDa) and the toxicity decreased with increasing substitution of LA. The transfection efficiency of different complexes was evaluated by luciferase assay. Compared with PEI (25 kDa) and NSC-g-PEI/DNA, NSC-g-PEI-LA showed good transfection activity and cell specificity to HepG2 cells. The results suggested that NSC-g-PEI-LA has the potential to be used as a safe and effective targeting gene vector.

Cell to cell transfer of the chromatin-packaged human β-globin gene cluster

Cell type-specific gene expression is regulated by chromatin structure and the transcription factors provided by the cells. In the present study, we introduced genes packaged into chromatin into target cells using a human artificial chromosome (HAC) and analyzed regulation of gene expression. The human β-globin gene cluster was built into an HAC (globin-HAC) and introduced into mouse embryonic stem (ES) cells using microcell-mediated chromosome transfer (MMCT); the adult-type human β-globin gene was expressed in bone marrow and spleen cells of the transgenic mice. In vitro differentiation of ES cells into mouse erythrocytes indicated that the natural sequential expression of ε, γ and β-globin genes was reproduced on the globin-HAC. Combination of MMCT and a novel chromosome transfection technique allowed transfer of globin-HAC from HT1080 cells into the human leukemia cell line K562, and from K562 cells back into HT1080 cells. Expression of the γ-globin gene, repressed in HT1080 cells, was activated in K562 cells without any processes of differentiation into adult erythroid cells, and was completely repressed again in HT1080 cells when transferred back from K562 cells. Thus, transfer of target genes packaged into chromatin using a HAC was useful for functional analyses of gene regulation.

Gene Modification of Human Primary Natural Killer Cells by Electroporation with mRNA or DNA Coding for An Anti-GD2 Chimeric Antigen Receptor.

Abstract 2467Poster Board II-444Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. The main restrictions to developing NK cells for immunotherapy are the limited quantity of cells available for adoptive transfer and their relative resistance to gene transfer by any method. We have developed an efficient method to expand CD3-CD56+ primary NK cells in vitro using K562 artificial APCs expressing membrane-bound IL21. Here we have investigated the potential of these expanded human NK cells to be gene modified through electroporation of DNA and mRNA. Expanded NK cells were electroporated (Amaxa Nucleofector device, program X-01) with DNA or mRNA coding for the GFP reporter gene, and expression of the transgene was evaluated by flow cytometry. Analysis at 48 hours post electroporation revealed that the viability of NK cells electroporated with GFP mRNA was 78% and those electroporated with GFP DNA was 69%. When electroporated with DNA, 32% of the viable NK cells were positive for GFP but had heterogeneous expression level, whereas 98% of viable cells were positive for GFP following mRNA electroporation, with much more homogeneous GFP expression (Figure).Based on this success we further investigated the potential of expanded NK cells to be gene modified with a Sleeping Beauty transposon/transposase vector system carrying the transgene for a second-generation Chimeric Antigen Receptor (CAR) against the GD2 ganglioside antigen, with signaling via the CD28 and CD3z endodomains. The GD2 antigen is abundantly expressed in neuroblastoma and melanoma and is therefore a relevant target for adoptive immunotherapy. Electroporation of expanded NK cells with the GD2-CAR transposon alone yielded 25% electroporation efficiency, with a viability of 55%. Electroporation of expanded NK cells with the GD2-CAR transposon and the transposase plasmid decreased the transfection efficiency to 14%. Nonetheless, expanded NK cells modified with the GD2-CAR showed improved killing of the target cell CHP-134 using the calcein AM release assay, as compared to unelectroporated expanded NK cells from the same donor. While freshly isolated human NK cells are highly resistant to gene modification, in this study we show that expanded human NK cells can be efficiently electroporated with both DNA and mRNA. NK cells modified with DNA to express CAR gain improved cytotoxic function against target cells, but viability and gene transfer efficiency are low. Since electroporation of GFP mRNA resulted in increased transduction efficiency and viability, we are now evaluating electroporation of expanded NK cells with GD2-CAR mRNA. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Gene Transfer Targeting Hepatocytes Using the PiggyBac Transposon System: An Approach towards Hemophilia A Correction and Long Term Expression of Factor VIII.

Abstract 3575Poster Board III-512Human Factor VIII (hFVIII) deficiency offers advantages as a disease target for gene therapy as small increases in factor VIII levels will alter the bleeding phenotype. In addition, both mouse and dog models of the disease are available for preclinical studies. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a “cut and paste” mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBAC can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Wu et al and others reported that piggyBAC transposase is highly efficient at catalyzing transposition in mammalian cells in vitro (PNAS 103: 15008-15013, 2006). To date, there are no published reports of in vivo gene transfer to mammalian livers using the piggyBAC transposon system. Advantages of this novel nonviral vector system include a large transgene cassette capacity, ease of production and purification, and the ability to excise itself precisely without leaving a footprint. We hypothesize that a piggyBAC transposon vector carrying a reporter gene cassette or the human FVIII cDNA along with a codon-optimized (co-) transposase will confer persistent gene expression and correction of the hemophilia A bleeding phenotype with the FVIII cDNA. PiggyBAC transposons were engineered to carry a hygromycin resistance gene (Hygro), a luciferase expression cassette (PB luciferase), or a human alpha1 antitrypsin reporter (hAAT). We evaluated co- transposase-mediated transposition in the Huh-7 human hepatoma cell line to verify function in hepatocytes. Using the PB hygro vector, we demonstrated that the co- transposase generated higher transposition efficiency than an inactive mutant in hepatocytes. We then showed in vivo persistence following hydrodynamic tail-vein injection using firefly luciferase expression driven by the murine albumin enhancer/human alpha anti-trypsin promoter. Luciferase expression measured via in vivo bioluminescence imaging persisted up to eight months in C57Bl/6 liver (duration of experiment). Following partial hepatectomies at 5 months post injection, expression was observed only in animals receiving PB luciferase transposon and an active transposase while expression in those treated with the inactive mutant dropped to background levels supporting that expression was from integrated transgene. We furthered these experiments by introducing PB hAAT via hydrodynamic tail-vein injection as before at either a low (12.5 micrograms each transposon and transposase) or high (50 micrograms each) dose. Serum hAAT levels were measured at 421ng/ml and 365ng/ml via ELISA at 3 months post-injection, respectively. PB vectors encoding hFVIII have been prepared, and our studies with these vectors are ongoing. These data represent one of the first studies to show persistent transgene expression in vivo from piggyBAC transposon gene transfer. Disclosures:No relevant conflicts of interest to declare.

Sustained transgene expression despite T lymphocyte responses in a clinical trial of rAAV1-AAT gene therapy

Alpha-1 antitrypsin (AAT) deficiency is well-suited as a target for human gene transfer. We performed a phase 1, open-label, dose-escalation clinical trial of a recombinant adeno-associated virus (rAAV) vector expressing normal (M) AAT packaged into serotype 1 AAV capsids delivered by i.m. injection. Nine AAT-deficient subjects were enrolled sequentially in cohorts of 3 each at doses of 6.9 x 10(12), 2.2 x 10(13), and 6.0 x 10(13) vector genome particles per patient. Four subjects receiving AAT protein augmentation discontinued therapy 28 or 56 days before vector administration. Vector administration was well tolerated, with only mild local reactions and 1 unrelated serious adverse event (bacterial epididymitis). There were no changes in hematology or clinical chemistry parameters. M-specific AAT was expressed above background in all subjects in cohorts 2 and 3 and was sustained at levels 0.1% of normal for at least 1 year in the highest dosage level cohort, despite development of neutralizing antibody and IFN-gamma enzyme-linked immunospot responses to AAV1 capsid at day 14 in all subjects. These findings suggest that immune responses to AAV capsid that develop after i.m. injection of a serotype 1 rAAV vector expressing AAT do not completely eliminate transduced cells in this context.

SP156 – Targeted gene transfer into facial muscles by pulsed laser

SP156 – Targeted gene transfer into facial muscles by pulsed laser

Endogenous HIV-1 Vpr-mediated apoptosis and proteome alteration of human T-cell leukemia virus-1 transformed C8166 cells

HIV-1 viral protein R (Vpr) can induce cell cycle arrest and cell death, and may be beneficial in cancer therapy to suppress malignantly proliferative cell types, such as adult T-cell leukemia (ATL) cells. In this study, we examined the feasibility of employing the HIV-vpr gene, via targeted gene transfer, as a potential new therapy to kill ATL cells. We infected C8166 cells with a recombinant adenovirus carrying both vpr and GFP genes (rAd-vpr), as well as the vector control virus (rAd-vector). G(2)/M phase cell cycle arrest was observed in the rAd-vpr infected cells. Typical characteristics of apoptosis were detected in rAd-vpr infected cells, including sub-diploid peak exhibition in DNA content assay, the Hoechst 33342 accumulation, apoptotic body formation, mitochondrial membrane potential and plasma membrane integrity loss. The proteomic assay revealed apoptosis related protein changes, exhibiting the regulation of caspase-3 activity indicator proteins (vimentin and Rho GDP-dissociation inhibitor 2), mitochondrial protein (prohibitin) and other regulatory proteins. In addition, the up-regulation of anti-inflammatory redox protein, thioredoxin, was identified in the rAd-vpr infected group. Further supporting these findings, the increase of caspase 3&7 activity in the rAd-vpr infected group was observed. In conclusion, endogenous Vpr is able to kill HTLV-1 transformed C8166 cells, and may avoid the risks of inducing severe inflammatory responses through apoptosis-inducing and anti-inflammatory activities.

Engineering the Surface Glycoproteins of Lentiviral Vectors for Targeted Gene Transfer

INTRODUCTIONVectors derived from retroviruses such as lentiviruses and oncoretroviruses are especially suitable tools for long-term gene transfer, because they allow stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors are preferred over vectors derived from oncoretroviruses such as murine leukemia virus (MLV) vectors, because they can transduce nonproliferating target cells. Moreover, lentiviral vectors that can target tissues specifically will be valuable for various gene-transfer approaches in vivo. To achieve targeted gene transfer, two types of surface modifications have been made to lentiviral vectors: (1) heterologous viral glycoproteins have been incorporated to exploit the tropism of other viruses (this is called pseudotyping), and (2) heterologous polypeptides have been fused to viral glycoproteins to retarget the lentiviral particles to a cell of interest. This article provides an overview of innovative approaches to upgrade lentiviral vectors for tissue targeting.

Hepatitis B virus envelope as a targeting gene transfer vector for hepatic cancer cells

Objective The aim of the study was to observe the transfection efficacy of hepatitis B virus envelope (HBVE) and evaluate its ability as a gene transfer vector for liver cancer cells.

Development of a Genetically Engineered Biomimetic Vector for Targeted Gene Transfer to Breast Cancer Cells

A biomimetic vector was genetically engineered to contain at precise locations (a) an adenovirus mu peptide to condense pDNA into nanosize particles, (b) a synthetic cyclic peptide to target breast cancer cells and enhance internalization of nanoparticles, (c) a pH-responsive synthetic fusogenic peptide to disrupt endosome membranes and facilitate escape of the nanoparticles into the cytosol, and (d) a nuclear localization signal from human immunodeficiency virus for microtubule mediated transfer of genetic material to the nucleus. The vector was characterized using physicochemical and biological assays to demonstrate the functionality of each motif in the vector backbone. The results demonstrated that the vector is able to condense plasmid DNA into nanosize particles (<100 nm), protect pDNA from serum endonucleases, target ZR-75-1 breast cancer cells and internalize, efficiently disrupt endosome membranes, exploit microtubules to reach nucleus and mediate gene expression. The therapeutic potential of the vector was evaluated by complexing with plasmid DNA encoding TRAIL (pTRAIL) and transfecting ZR-75-1 cells. The results demonstrated that up to 62% of the ZR-75-1 breast cancer cells can be killed after administration of pTRAIL in complex with the vector.

Targeting Heme Oxygenase

Heme oxygenase (HO) is important in attenuating the overall production of reactive oxygen species through its ability to degrade heme and to produce carbon monoxide, biliverdin/bilirubin, and release of free iron. Excess free heme catalyzes the formation of reactive oxygen species, which leads to endothelial cell (EC) dysfunction as seen in numerous pathologic vascular conditions including systemic hypertension and diabetes, as well as in ischemia/reperfusion injury.The up-regulation of HO-1 can be achieved through the use of pharmaceutical agents such as metalloporphyrins and statins. In addition, atrial natriuretic peptide and nitric oxide donors are important modulators of the heme-HO system, either through induction of HO-1 or the increased biologic activity of its products. Gene therapy and gene transfer, including site- and organ-specific targeted gene transfer have become powerful tools for studying the potential role of the 2 isoforms of HO, HO-1/HO-2, in the treatment of cardiovascular disease, as well as diabetes. HO-1 induction by pharmacological agents or the in vitro gene transfer of human HO-1 into ECs increases cell cycle progression and attenuates angiotensin II, tumor necrosis factor-alpha, and heme-mediated DNA damage; administration in vivo corrects blood pressure elevation after angiotensin II exposure. Delivery of human HO-1 to hyperglycemic rats significantly lowers superoxide levels and prevents EC damage and sloughing of vascular EC into the circulation. In addition, administration of human HO-1 to rats in advance of ischemia/reperfusion injury considerably reduces tissue damage.The ability to up-regulate HO-1 either through pharmacological means or through the use of gene therapy may offer therapeutic strategies for the prevention of cardiovascular disease in the future. This review discusses the implications of HO-1 delivery during the early stages of cardiovascular system injury or in early vascular pathology, and suggests that pharmacological agents that regulate HO activity or HO-1 gene delivery itself may become powerful tools for preventing the onset or progression of various cardiovascular diseases.