Flt-1 is a high-affinity, VEGF tyrosine kinase cell surface receptor found almost exclusively on endothelial cells 94 , , Alternative splicing of Flt-1 pre-messenger RNA mRNA generates two distinct products, one encoding the full-length membrane-spanning receptor and a second encoding a soluble form sFlt that is made up of only six of the seven immunoglobulin sequences in the extracellular domain without the transmembrane and the intracellular domains , Although both species bind to VEGF with similar affinity, binding to the sFlt form does not lead to signal transduction because it is not cell associated and lacks the intracellular tyrosine kinase domains , Angiostatin and endostatin.

Both angiostatin and endostatin are two secreted proteins that may play a role in maintaining the quiescent state of normal endothelial cells. Angiostatin is a kd protein whose sequence is identical to that of the first four kringle structures of plasminogen 58 , , , Angiostatin is cleaved by elastase from plasminogen The two molecules have different biologic functions: Neither plasminogen nor plasmin inhibits angiogenesis, and angiostatin has no anticoagulant activity Angiostatin acts specifically on endothelial cells without affecting tumor cells directly, and administration of angiostatin to tumor-bearing mice leads to an inhibition of angiogenesis and an increased apoptotic rate in the tumor cells, resulting in a state of tumor dormancy Analogous to angiostatin, endostatin is an kd protein that is cleaved enzymatically from collagen XVIII It has antiangiogenesis activity similar to that of angiostatin Since endothelial cells are the primary structural units of blood vessels, the signals that initiate angiogenesis do so by interacting with receptors on endothelium 86 ,98— Inherent in the strategy of using gene therapy to suppress angiogenesis of tumors is that the positive and negative regulators of angiogenesis act on the endothelial cells in trans ; i.

This is important for strategizing antiangiogenesis gene therapy, because it eliminates the challenge of having to deliver the antiangiogenesis gene to a specific cell type; i. These considerations provide the biologic basis for the use of gene transfer strategies to achieve regional antiangiogenesis; i.

Thus, in vivo transfer of the antiangiogenesis gene to normal and malignant cells within the target organ should result in a secretion of the therapeutic protein into the extracellular milieu by both cell populations.

This common extracellular pool of secreted antiangiogenesis factors can then act on the endothelial cells in trans to dampen angiogenesis.

The net result is therapeutic antiangiogenesis without the need to transduce every cell in the organ or the need to target any cell population specifically.

This advantage is important because the current gene transfer technology is limited by the inability to deliver the therapeutic gene to every target cell in vivo Various proof-of-principle experimental animal studies suggest that gene therapy may be an effective means to deliver antiangiogenesis therapy to solid tumors Table 4.

These antiangiogenesis gene therapy strategies can be categorized into those that suppress the proangiogenic signal and those that augment the inhibition of angiogenesis. The proangiogenic signal can be suppressed by decreasing the amounts of the angiogenic mediator available to induce tumor neovascularization or by interfering with the process of the angiogenic mediator signaling within the endothelial cell.

This strategy capitalizes on the ability of gene therapy to alter the genetic repertoire of target cells—in this case, the tumor cells overexpressing specific angiogenic mediators.

The fundamental approach is to transfer antisense sequences or ribozymes that will deplete mRNA coding for the angiogenic mediator. The proof-of-principle study demonstrating that such an approach is feasible capitalized on the knowledge that VEGF is a potent angiogenic mediator secreted by many tumor types.

By using an antisense construct against VEGF, Cheng et al. One challenge to this approach is that antisense functions as a cis effect only. Thus, for this antisense gene therapy to work in vivo , a large percentage of cells needs to be inhibited. This approach is based on the concept that interfering with the normal function of receptors for angiogenic mediators should potently disrupt the angiogenesis cascade.

To evaluate this concept, Millauer et al. The mutant lacks the intracellular domain but retains the extracellular and the transmembrane domain; as such, the mutant receptor remains cell associated but dysfunctional.

By infecting endothelial cells with the retrovirus vector coding for the mutant receptor, heterodimerization occurs between the mutant receptor and the full-length, native Flk-1 receptor on endothelial cell membrane.

Unlike the native homodimeric Flk-1 receptor, the heterodimer was unable to bring about signal transduction and endothelial cell activation. As a proof of principle, a combined ex vivo and in vivo strategy was used, where an ecotropic packaging cell line producing a retrovirus vector coding for the mutant receptor was co-implanted with glioblastoma cells in nude mice; the result was suppression of tumor growth compared with findings in control animals It is interesting that intratumoral administration of the retroviral supernatant also suppressed growth of primary glioblastomas.

To accomplish this, an adenovirus vector Adsflt was designed to deliver a cDNA coding for a truncated form of the Flt-1 VEGF receptor that lacked the intracellular domain, the transmembrane domain, and part of the extracellular domain.

The product expressed by the vector sFlt is a diffusible, soluble receptor molecule that can bind to VEGF molecules with high affinity. Irrespective of the responsible mechanism, when the Adsflt vector was administered in vivo to mice bearing primary or metastatic tumors that arose from syngeneic colon carcinoma cells, substantial tumor suppression was observed, and the treated animals had a statistically significant survival advantage.

Importantly, the therapeutic effect was found to be regional, i. Since angiogenesis is the net result of a dynamic balance between the proangiogenic and antiangiogenic factors in the extracellular microenvironment of the tumor, increasing the local concentrations of endogenous inhibitors of angiogenesis should shift the balance between angiogenesis and antiangiogenesis in favor of the latter.

To evaluate this concept, Tanaka et al. Importantly, the growth of gliomas in nude mice could be suppressed following intratumoral administration of the vectors; the treated tumors appeared hypovascular, and the treated animals had a survival advantage over the control animals In another study evaluating the concept of increasing the local concentration of inhibitors of angiogenesis 59 , the TSP-1 cDNA was transfected into breast carcinoma cells, followed by their injection into the mammary fat pads of nude mice.

The resulting tumors were found to be smaller and to have a lower metastatic potential than the naive i. Although the gene therapy approach to antiangiogenesis therapy for solid tumors is still in its infancy, the preliminary data developed to date suggest gene therapy should live up to its theoretical potential of providing high, local concentrations of the therapeutic molecule, while avoiding the potential toxicity of systemic administration.

However, there are several challenges that will have to be overcome before antiangiogenesis gene therapy becomes a useful strategy to treat human tumors.

Some of these challenges are specific for gene therapy per se ; others are generic challenges for antiangiogenesis. It is apparent from experimental animal studies and from the early clinical studies of systemic antiangiogenesis therapy that, for antiangiogenesis therapy to be effective in treating tumors, the antiangiogenesis effects must be maintained for a long time 20 , If we assume that remaining tumor cells have the potential to express proangiogenic mediators, interruption of antiangiogenesis therapy has the potential risk of tipping the angiogenesis balance in favor of proangiogenesis, allowing the tumor to emerge from its dormancy.

A successful gene therapy for antiangiogenesis should therefore have a sustained effect. This is an important challenge for current gene transfer vectors, which either inherently provide only transient expression e.

There are various solutions to the challenge of maintaining persistent expression of a transgene following gene transfer.

They include the following: 1 designing vectors to be more efficient in entering the target cell and transferring genes to the nucleus, 2 permanently incorporating the transgene into the target cell genome, 3 designing the vector to be stealthy with regard to detection by the hosts' innate and adaptive immune systems, 4 using pharmacologic agents to suppress host responses to the vectors, 5 designing the transgene to code for an antiangiogenic protein that has a longer biologic half-life in the target organ, and 6 using promoters that are resistant to shutdown by the host cell.

Although there are risks to this approach in that the other antitumor gene strategy may eliminate the cells expressing the antiangiogenesis genes, the initial tumor reduction brought about by an antiangiogenesis treatment might be maintained by chronic tumor suppression from antitumor genes that function to suppress tumor growth by other mechanisms.

In this context, gene therapy-based immunotherapy may be the best choice for combined therapy, in that it would provide tumorspecific suppression, while the gene therapy-based antiangiogenesis therapy would attack the tumor by a very different mechanism, independent of the cell site of the antiangiogenesis genes.

While the local production of a therapeutic antiangiogenesis protein is an inherent advantage of gene therapy in that it limits the risk of promiscuous systemic antiangiogenesis, such a strategy suffers from an inability to treat widespread metastases.

Thus, one challenge of future antiangiogenesis gene therapy is to target the vector or its transgene product to tumor-associated vessels, permitting systemic treatment of disseminated tumors. Such a goal may be achieved when more is known about the phenotypic differences between normal vessels and those that are induced by and support growing tumors.

In this regard, there are emerging data suggesting that tumor neovasculature behaves differently from its normal counterpart in that, although the tumor neovasculature is composed of normal cells, its architecture is abnormal. For example, tumor blood vessels are leaky and aberrantly arranged, with unusual fan and spiral motifs, forming right angles and arteriovenous shunts 15 , Up-regulation of α v β 3 integrins is also a feature of new blood vessels in tumors, a biologic process believed to be critical for the survival and differentiation of vascular cells undergoing angiogenesis Since adenovirus vectors use α v β 3 integrins as an internalization signal, it may be possible to capitalize on this feature to design adenovirus vectors specific for active angiogenesis, such as occurs in growing tumors — Another possible target to achieve tumor vessel specificity is E-selectin One of the challenges to success of antiangiogenesis gene therapy is to ensure that the strategy will be applicable to a broad range of tumor types, regardless of their profiles of angiogenic mediators.

A successful strategy will have to counteract the proangiogenic phenotype induced by VEGF, bFGF, and likely other angiogenic mediators. It may therefore be more rational to target genes that express products that function to interrupt processes downstream in the angiogenesis cascade.

In this regard, gene therapy approaches that target the common signaling cascades in the end organ of angiogenesis, i. With a better appreciation of the critical and universal dependence of tumor progression on neovascularization, it is rational to hypothesize that suppression of this rate-limiting step could suppress the growth of a wide range of tumor types.

As the cellular and molecular events that underlie tumor angiogenesis become better defined, rational strategies can be derived to apply this molecular tourniquet. An ideal antiangiogenesis strategy should be targeted to only the organs that contain the tumors and should not interfere with normal angiogenesis; it must achieve a high ratio of regional-to-systemic concentrations to minimize systemic toxicity; it must have a biologic half-life sufficient to counter the proangiogenesis phenotype of the tumor; and its antiangiogenesis effects should be regulatable.

Gene transfer strategies potentially satisfy many of these requirements. Critical to the success of antiangiogenesis gene transfer is the fact that endothelial cells are activated or suppressed in trans , depending on the composition of the extracellular milieu. In this context, gene therapy for angiogenesis does not have to transduce all or any specific populations of cells in the target organs to achieve a high, local concentration of the antiangiogenesis proteins.

Antiangiogenesis treatment is likely to be most effective in a low tumor burden state. In such a setting, therapeutic antiangiogenesis can be expected to prolong the state of tumor dormancy by suppressing micrometastases that remain despite successful treatment of the primary tumors.

One appropriate clinical approach to using antiangiogenesis therapy in cancer is to combine it with conventional therapy to reduce the initial tumor burden, followed by its use in an adjuvant setting to prolong disease-free survival.

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Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Why Use Gene Therapy to Deliver Antiangiogenesis Agents?

Biology of Tumor Angiogenesis Relevant to Antiangiogenesis Gene Therapy. Antiangiogenesis With the Use of Gene Therapy. Challenges to Successful Antiangiogenesis Gene Therapy.

Journal Article. Gene Therapy Strategies for Tumor Antiangiogenesis. Hwai-Loong Kong , Hwai-Loong Kong. Oxford Academic.

Ronald G. Revision received:. Split View Views. Cite Cite Hwai-Loong Kong, Ronald G. Select Format Select format. ris Mendeley, Papers, Zotero. enw EndNote. bibtex BibTex. txt Medlars, RefWorks Download citation.

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Abstract Based on the concept that solid tumors cannot grow without the generation of new blood vessels, there is growing interest in the use of antiangiogenesis agents to inhibit tumor growth.

Open in new tab Download slide. Google Scholar Crossref. Search ADS. Google Scholar PubMed. OpenURL Placeholder Text. Review article: angiogenesis, neovascular proliferation and vascular pathophysiology as targets for cancer therapy. Google Scholar Google Preview OpenURL Placeholder Text.

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Google Scholar OpenURL Placeholder Text. Also, as a new targeted strategic approach, nanotechnology medical applications have been intensively studied to deliver anti-angiogenic drugs into the tumoral specific sites using nanomaterials as cerium oxide, gold, silver, copper, silica, based on carbon or hyaluronic acid and others Mukherjee and Patra, Besides their role in normal tissue maintenance, angiogenesis initiation may indicate a shift from tumor latency to malignant active growth and recurrence of the disease.

The precise functions of pro- and anti-angiogenic factors and the interactions between them in tumor angiogenesis are not fully understood and the important question is how anti-angiogenic medicine can be improved.

However, the mechanisms of induction of vascularization and subsequent development from precancerous lesions to micrometastases achieved by angiogenic strategies for vessel recruitment are not yet fully elucidated in all pathological cases.

Specific agents that can block tumor vascularization are required to inhibit angiogenesis and tumor growth. This review summarizes angiogenic factors involved in each step of vessel development to present an integrated overview of tumor vascularization models such as cooption, intussusception, sprouting angiogenesis, vasculogenic mimicry, and angioblasts which, depending on the context, can be helpful for targeted or combined anti-angiogenic therapies.

Moreover, we present the epigenetic changes in cancer which in contrast with genetic changes, are potentially reversible, increasing the prospect that epigenetic therapy will be able to mediate tumor fate.

In addition to more disease-specific biomarkers, an important issue remains optimization of the dose and frequency of delivery of anti-angiogenic drugs. Current efforts for biomarker discovery in cancer have primarily focused on multi-gene expression patterns, but complementary analysis of DNA methylation signatures may lead to diagnostic and prognostic improvement and better cancer therapy strategies.

The major limitations of drug delivery systems remain the lack of specificity. However, drug-specific therapies that use a lower dose of epi-drugs could improve the effectiveness and tolerability of treatments. Another approach that might improve cancer therapy is the optimization of the dose and duration of release of anti-angiogenic drugs, with potential to alleviate colateral damage that conventional treatments that are toxic to both tumor and normal cells might produce.

Future directions for these treatments may include combined drug delivery systems that might target several types of anti-angiogenic factors for synergistic or additive therapeutic effects, and might increase the efficacy and specificity along with reduction of side effects.

VA and CD were involved in study conception. IS and CB were involved in study design. VA wrote the manuscript with support from IS, CB, and CD. All authors reviewed and approved the final version of the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

We gratefully acknowledge the funding from the project Competitiveness Operational Programme COP A1. Adair, T. Chapter 1, Overview of Angiogenesis.

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The simplest non-viral gene delivery system uses naked expression vector DNA. Direct injection of free DNA into certain tissues, particularly muscle, has been shown to produce surprisingly high levels of gene expression, and the simplicity of this approach has led to its adoption in a number of clinical protocols.

However, naked DNA and peptides have a very short half life due to in vivo enzymatic degradation. Plasmid DNA suffers from low transfection efficiency. However, there are reports showing efficacy of naked plasmid DNA administration.

Intra-tumoral administration of naked plasmid DNA encoding mouse endostatin inhibits renal carcinoma growth [ ]. Intramuscular administration of the endostatin gene could significantly retard the growth of metastatic brain tumors [ ].

A single intramuscular administration of the endostatin gene could secret endostatin for up to 2 weeks and could inhibit systemic angiogenesis [ ]. Injected endostatin gene also inhibited both the growth of primary tumors and the development of metastatic lesions.

Thus, these results demonstrate the potential utility of intramuscular delivery of an antiangiogenic gene for treatment of disseminated cancers. However, in comparison to naked DNA, complexes of plasmid DNA with liposomes are relatively more stable with higher potency for transfection [ ].

Liposomes are microscopic spherical vesicles of phospholipids and cholesterol. Recently, liposomes have been evaluated as delivery systems for drugs and have been loaded with a great variety of molecules such as small drug molecules, proteins, nucleotides and even plasmids.

The advantages of using liposomes as drug carriers are that they can be injected intravenously and when they are modified with lipids which render their surface more hydrophilic, their circulation time in the bloodstream can be increased significantly.

They can be targeted to the tumor cells by conjugating them to specific molecules like antibodies, proteins, and small peptides [ ].

The cationic liposomes can significantly improve systemic delivery and gene expression of DNA [ ]. Systemic, liposome-mediated administration of angiostatin could suppress the growth of melanoma tumors in mice [ ]. Similar findings were observed by Chen and co-workers with angiostatin and endostatin [ ].

It has been shown that angiogenic ECs take fold more cationic liposome:DNA complexes than corresponding normal ECs. Thus, preferential uptake of cationic liposomes could be used to target diagnostic or therapeutic agents selectively to angiogenic blood vessels in tumors [ ].

Liposomes modified with angiogenic homing peptide for ECs can strongly suppress tumor growth compared to unmodified liposomes [ ].

Janssen et al showed that the coupling of cyclic RGD-peptides to the surface of PEG-liposomes can target to tumor endothelium [ ]. Tumor vessel-targeted liposomes can also be used to efficiently deliver therapeutic doses of chemotherapy [ ].

Antisense oligodeoxynucleotides ODNs are synthetic molecules that block mRNA translation. They can be used as a tool to inhibit mRNA translation of a diseased gene. There are reports demonstrating use of VEGF and VEGFR antisense RNA in preclinical models.

Angiogenesis and tumorigenicity as measured by MVD and tumor volume, respectively of human esophageal squamous cell carcinoma can be effectively inhibited by VEGF antisense RNA [ ].

Im and co-workers used an adenovirus to transfer antisense VEGF sequence into glioma cells in vitro and in vivo [ ]. The treatment resulted in reduction of the level of the endogenous VEGF mRNA and protein and inhibited growth of glioma tumors.

VEGF mediated neovascularization can also be inhibited by combination of antisense oligonucleotides to VEGFR1 and VEGFR2 [ ]. Expression of antisense RNA to Ang1 could reduce tumor volume, decrease MVD and increase apoptosis in nude mice [ ].

Expression of antisense to integrin subunit beta 3 inhibits microvascular EC capillary tube formation in fibrin, with the extent of down-regulation correlating with the extent of tube formation inhibition [ ].

The ability of small dsRNA to suppress the expression of a gene corresponding to its own sequence is called RNA interference RNAi.

The discovery of RNAi has added a promising tool to the field of molecular biology. Introducing the SiRNA corresponding to a particular gene will knock out the cell's own expression of that gene. The application of SiRNA to silence gene expression has profound implications for the intervention of human diseases including cancer.

There are published reports using SiRNA to silence expression of angiogenic genes. SiRNA targeted to either subunit of the alpha6beta4 a laminin adhesion receptor integrin reduced its cell surface expression and resulted in decreased invasion of MDA-MB breast carcinoma cells [ ].

The disadvantage to simply introducing dsRNA fragments into a cell is that gene expression is only temporarily reduced. However, Brummelkamp et. developed a new vector system, named pSUPER, which directs the synthesis of siRNA in mammalian cells [ ].

The authors have shown that siRNA expression mediated by this vector causes persistent and specific down-regulation of gene expression, resulting in functional inactivation of the targeted gene over longer periods of time.

VEGF carries out multifaceted functions in tumor development. DNA-vector based RNAi, in which RNAi sequences targeting VEGF isoforms, has potential applications in isoform-specific knock-down of VEGF [ ].

However, the disadvantages of non viral vectors — stability, non-specific uptake by various tissues, poor adsorption, short half life in the circulation, aggregate formation, and low in-vivo potency for cell transfection — continue to limit its use.

The main characteristics of any viral vector are easy purification into high titers, to mediate targeted gene delivery and prolonged gene expression with minimal side effects. There are 5 main classes of clinically applicable viral vectors; adenoviruses, adeno-associated viruses AAVs , retroviruses, lentiviruses, and herpes simplex-1 viruses HSV-1s [ ].

The main difference between these vectors is retroviruses and lentiviruses can integrate into the genome, whereas the other three classes predominantly persist as extrachromosomal episomes.

Although the advantage of chromosomal integration is long term transgene expression, these vectors can infect dividing cells only. The non-integrated viral vectors can infect non-dividing cells, but are not a favorable choice to bring about stable genetic change. Adenovirus is a double stranded DNA virus.

It binds initially to the target cell through the viral fiber protein; however, the subsequent cell entry involves interaction between the viral capsid penton proteins and integrins on the target cell.

Adenoviruses can be produced in high titers and can efficiently deliver the therapeutic gene. Replication-defective adenoviral vectors are indeed particularly well suited for cancer gene therapy as they lead to a transient, but robust, expression of the transgene, and efficient in vivo gene transfer has been reported especially in the liver after systemic injection.

However, they do not integrate into the host genome and the gene expression is transient. Also, adenovirus elicits an immune response resulting in an elimination of vector expressing cells. Despite these apparent drawbacks, the adenovirus remains a popular vector for gene therapy due to its high gene transfer efficiency and high level of expression in a wide variety of cell types.

Adenovirus can be targeted to pulmonary endothelium by complexing it with a bispecific antibody to viral particle and angiotensin-converting enzyme [ ]. Bilamellar cationic liposomes can also be used to encapsulate adenovirus. The encapsulated adenovirus can transduce CAR negative cells and is resistant to the neutralizing anti-adenoviral antibodies, allowing the readministration of the adenovirus [ ].

Retroviruses are a class of enveloped viruses containing a single stranded RNA molecule as the genome. Retrovirus vectors have been used in the majority of human gene transfers.

They are able to efficiently integrate permanently into the human genome where they provide the basis for permanent expression of up to 8—9 kb of foreign DNA. Simple retroviruses, such as murine leukemia virus MLV , and the vectors derived from them, require cell division for infection and thus possess a degree of inherent specificity for the rapidly dividing cells of neoplastic tissue [ ].

Though transgene expression is usually adequate in vitro, prolonged expression is difficult to attain in vivo. Also, retroviruses are inactivated by complement proteins and inflammatory IFN, specifically IFN-alpha and IFN-gamma [ , ].

A major shortcoming of retrovirus-derived vectors is their tendency to revert to replication-competent retrovirus RCR , which could lead to fatal neoplasms. With the use of the latest packaging cell lines and vectors, the risk of RCR-generation has been drastically reduced.

Currently, the greatest safety concern of using retroviral vectors is related to the risk of malignant transformation following oncogene activation due to random retroviral genomic integration and will be discussed in more detail below.

The adeno-associated virus AAV is a vector that combines some of the advantages of both the adenoviral and retroviral vectors. It can efficiently transfer genes to a number of different cell types.

The broad host range, low level of immune response, and longevity of gene expression observed with these vectors has enabled the initiation of a number of clinical trials using this gene delivery system [ ].

A potential barrier, however, is the low transduction efficiencies of recombinant AAV vectors. AAV tropism can be genetically engineered by use of phage display-derived peptides to generate vectors that are selective for the vasculature [ ]. The journal Nature recently reported concerns that there is a possibility that recombinant AAV vectors may cause or contribute to cancer in gene therapy subjects [ ].

However, because of infrequent integration efficiency of AAV, the risk of cancer in current AAV trials is negligible [ ].

Lentiviral vectors represent a new vector system that can achieve permanent integration of the gene into non-dividing cells. Gene transfer can be achieved in very quiescent cells, nondividing or terminally differentiated cells such as neurons. Lentiviral vectors are especially useful in transducing cells which lack receptors for adenoviruses.

A broad tissue tropism for lentivirus can be achieved using variety of viral envelopes [ ]. So far, lentiviral vectors expressing matrix metalloproteinase-2 MMP-2 , angiostatin and endostatin have been developed [ , ]. However, lentivirus has a low transduction efficiency for ECs and may result in significant vector-associated cytotoxicity [ ].

The herpes simplex virus HSV thymidine kinase gene tk therapy with ganciclovir forms the basis of a widely used strategy for suicide gene therapy [ ]. HSV can also be used to deliver a therapeutic gene of interest, especially to the nervous system. The advantages in using HSV include its wide host range, its ability to accommodate large genes, and its ability to establish long-lived asymptomatic infections in neuronal cells.

However, the virus's ability to replicate lytically in the brain, under some circumstances causing encephalitis, has led to fears about its potential safety for ultimate use in humans. The systemic delivery of viral vectors can lead to non specific uptake by various different tissues and hence result into systemic toxicity because of transgene expression.

Local delivery can circumvent this problem; however, many times the tumor is not accessible for local injection. Hence, transgene expression in the targeted tissue is very important.

Targeting tumor vasculature by gene therapy represents an ideal target, as tumor blood vessels are easily accessible to systemically applied vectors. Certain considerations such as vascular dependency of a tumor, the differences between tumor associated and normal vasculature, and accessibility of tumor vasculature to circulating vectors must be addressed.

Targeted viral vectors can be constructed in several ways. The first approach is pseudotyping, in which one species of virus is made to incorporate the envelope protein of another virus.

Adeno-associated virus genome with its inverted terminal repeats can be packaged in the capsids of different serotypes which enables transduction with broad specificity [ ]. The second approach is to genetically modify the viral capsid protein to incorporate a small peptide coding for a specific receptor and hence, allow targeting by ligand-receptor internalization.

Tissue specific targeting can also be achieved by conjugating specific antibodies to receptors onto the viral capsids [ ]. Various transductional and transcriptional approaches have also been devised to improve targeting of adenoviral vectors. Using this approach, Reynolds et al increased the selectivity of transgene expression by , for lung versus liver, the usual site of vector sequestration [ ].

Another strategy is to use vectors guided by EC specific promoters, such as the promoters for endoglin, endothelin-1 gene, and von Willebrand factor. The murine preproendothelin-1 promoter is highly specific for ECs and could be use to achieve very high levels of gene expression in vascularized tumor metastases compared to normal or less vascularized primary tumor [ ].

Whereas most antiangiogenic agents prevent new blood vessel formation, vascular targeting agents VTAs destroy pre-existing blood vessels of solid tumors. VTAs can be more active in large tumors and produce a characteristic pattern of widespread central necrosis.

Combination of VTAs and angiogenic inhibitors can lead to synergistic effects. Two major types of VTAs are ligand-directed VTAs to deliver gene product to tumor endothelium and small molecule VTAs that exploit pathophysiological differences between tumor and normal endothelium [ ].

The ligand-directed VTAs use ligands specifically expressed on the tumor endothelium, eg. VEGF receptors, αvβ3 integrins, tissue factor, and cell adhesion molecules like VCAM A small molecule VTA described thus far is the tubulin binding agent combretastatin, which destabilizes the tubulin cytoskeleton.

It has antiproliferative and cytotoxic effects on both proliferating tumor and ECs. It results in extensive and prolonged shut-down of blood flow in established tumor blood vessels, with much less effect in normal tissues [ ]. Recently, bacteriophage vectors have been developed for targeted gene delivery of antiangiogenic VTAs.

Phage display represents a novel method of individually displaying up to tens of billions of peptides, proteins, including human antibodies and enzymes, on the surface of a small bacterial virus called a phage. Phage display allows producing and searching through large libraries of peptides and proteins to rapidly identify those compounds that bind with high affinity and high specificity to targets of interest.

Pasqualini and Ruoslahti first distinguished active proliferating microvascular ECs and quiescent nonproliferating ECs using an in vivo phage display technique [ ]. The phage display peptide libraries have been used to identify peptides that home to tumors through the circulation and that specifically bind to the tumor ECs or lymphatic cells [ , ].

Phage displaying an Arg-Gly-Asp RGD -containing peptide binds to alpha v integrins with high affinity and homes to tumors when injected intravenously into tumor-bearing mice. Phage displaying the cyclic peptide His-Try-Gly-Phe HWGF specifically targets angiogenic blood vessels in vivo and specifically inhibits MMP-2 and MMP-9 metalloproteinases [ ].

MMP-2 can directly bind to αvβ3 integrin on tumor cells [ ]. These tumor endothelial specific signatures were later described by Dr. Folkman as an angiogenic zip codes [ ]. The picture of integrins and their ligands is very complex. The αvβ3 integrin is expressed on proliferating ECs such as those present in growing tumors of various origins.

VEGF-induced EC migration requires interaction between VEGF receptor2 VEGFR2 and αvβ3 to drive the activation of downstream mitogenic pathways [ ]. Several studies have demonstrated that αvβ3 is involved in melanoma cell invasion and promotes metastasis reviewed in [ ].

Although the precise mechanisms of αvβ3-promoted tumor progression are not clear, various studies suggest roles for αvβ3 in selective tumor cell migration, generation of growth and survival signals, intracellular signaling and generation of MMPs [ ].

It has been observed that antibody to αvβ3 integrin can block angiogenesis without affecting the normal vasculature [ , ]. The αvβ3 integrin can also be blocked with small peptides containing Arg-Gly-Asp RGD amino acid sequence.

Exposure of human EC to TNF and interferon-γ decreases αvβ3-dependent EC adhesion and survival [ ]. Other integrins such as αvβ5, and αvβ1 are also considered very significant for the regulation of angiogenesis. The β3 chain play a significant role in promoting angiogenesis, although the lack of it can also promote angiogenesis, given that the angiogenesis inhibitor tumstatin binds through this receptor [ 41 ].

Also, the expression of integrin αvβ6, a fibronectin receptor, promotes migration and invasion in squamous carcinoma cells [ ]. Bacteriophages expressing ligands for specific receptors like integrins, growth factors, and antibody can be made to infect mammalian cells [ — ].

The phage vectors are an attractive alternative to existing animal viral vectors because they lack intrinsic tropism for mammalian cells and can be produced in bacteria in large titers.

Burg et al tested 14 human cancer cell lines from different tissues, showing viral transduction efficiency varies from 0. However the transduction efficiency can be improved substancially by treating cells with camptothecin [ ].

Thus, using specific targets in the tumor endothelium, it is possible to target gene therapy specifically to the tumor endothelium. In recent years, significant effort has been devoted to developing nanotechnology for drug delivery since it offers a suitable means of delivering small molecular weight drugs as well as macromolecules such as proteins, peptides or genes [ ].

Nanoparticles NPs are submicron-sized polymeric colloidal particles with a therapeutic agent of interest encapsulated within their polymeric matrix or adsorbed or conjugated onto the surface [ ]. NPs have in general relatively higher intracellular uptake because of their smaller size.

The therapeutic efficacy of the NPs could be due to their ability to protect the therapeutic agent from degradation due to lysosomal enzymes and sustained intracellular retention.

Nanoparticles conjugated to the contrast agent like gadolinium can be used in magnetic resonance based imaging techniques [ , ]. They can be targeted to tumor endothelium by covalently coupling to different ligands or antibodies [ ].

David Cheresh's group at the Scripps Research Institute showed that NPs coupled to an integrin αvβ3-targeting ligand can deliver genes selectively to angiogenic blood vessels in tumor-bearing mice with no detectable levels in the other tissues [ ].

Further, the authors showed that NPs conjugated to a mutant Raf gene blocks endothelial signaling and angiogenesis in response to multiple growth factors. Despite the tantalizing promise of gene therapy, there has been much recent alarm in the field and no review of the current state of and potential future role of gene therapy would be complete without noting the major current controversy in the field.

The first true successes of gene therapy in clinical trials were reported in and , in which scientists successfully treated children suffering from SCIDs by retrovirally transducing hematopoietic stem cells with the gamma-c gene [ 32 ]. Unfortunately, 2 of the 10 children subsequently developed leukemia-like conditions, eventually attributed to retroviral vector integration in proximity to the LMO2 proto-oncogene promoter, leading to aberrant transcription and expression of LMO2 [ ].

These cases garnered enormous attention from scientists, regulators and the general public [ ]. Reaction varied but some countries even imposed a general moratorium on trials involving retroviral gene transfer.

Though in most countries trials have now been allowed to resume, the reaction has thrown the field of gene therapy into recession and has discouraged many scientists from starting new clinical trials [ ]. As addressed above, the development of better vectors remains the cornerstone for advancing the field of antiangiogenic gene therapy of cancer.

Important concerns about oncogene activation by retroviral integrating vector systems remain. Also, currently available nonviral vectors are not very efficient.

Studies have shown advantages to combining antiangiogenic agents with each other, as well as with chemotherapy and radiation [ ]. Combining agents that target a number of different and synergistic pathways may yield improved antitumor effects and long term suppression of metastatic progression.

For example, the combination of SU, VEGFR2 tyrosine kinase inhibitor and low-dose endostatin reduced tumor growth more efficiently than monotherapy alone [ ]. Whereas SU specifically inhibits vascular endothelial growth factor signaling, low-dose endostatin is able to inhibit a broader spectrum of diverse angiogenic pathways directly in the endothelium.

Gene therapy strategies certainly need to be further evaluated in these combination settings. More specific to the field of antiangiogenic gene therapy, there is much enthusiasm for the role that antiangiogenic agents may play in preventive therapy.

Because antiangiogenic therapy is generally less toxic and less susceptible to acquired resistance, angiogenesis inhibitors have been considered an ideal prophylactic agent in patients at high risk for cancer or for recurrence of cancer [ 29 ].

At our institution, we are currently recruiting patients with previously resected recurrent or metastatic colorectal cancer and randomizing these patients to receive thalidomide or placebo.

Gene therapy offers an ideal strategy for long term, continuous production of antiangiogenic agents that may prevent development of primary or recurrent disease.

Additionally, many leaders in the field of angiogenesis now believe that some of the most important future cancer therapies may not completely eradicate all tumor cells in an individual but, instead, may turn cancer into a chronic manageable disease [ 32 ].

Gene therapy strategies leading to increased production of endogenous angiogenesis inhibitors would seem perfectly suited to support such an approach by tipping the balance toward a more antiangiogenic state. Enthusiasm for antiangiogenic approaches to cancer therapy has never been higher, given the recent approval by the FDA for the first angiogenesis inhibitor and given the recent encouraging clinical trial data [ ].

The versatility shown by the field of antiangiogenesis has enabled many human cancers to be realistically considered for therapeutic intervention and has considerably broadened the scope of gene therapy. Because of the difficulties and high costs of manufacturing numerous endogenous inhibitors of angiogenesis and because of the need for chronic administration of these agents, gene therapy remains an exciting strategy to circumvent these difficulties.

Although no clinical gene therapy trials to date have utilized a focused antiangiogenic strategy, an abundance of preclinical research demonstrates that gene therapy-based antiangiogenesis approaches are an effective means of controlling and even eradicating tumor growth in animal models.

As better vectors are developed, combination strategies continue to evolve, and increased understanding of the complex role that endogenous angiogenesis inhibitors play in tumor growth and progression takes place, antiangiogenic gene therapy will certainly be evaluated in future clinical trials.

Antiangiogenic Gene Therapy: Recent Developments. The figure depicts different modes of gene therapy directed to tumor endothelial cell EC and its microenvironment. The expression of EC specific cell surface molecules like, vascular endothelial growth factor receptor VEGFR , E selectin or angiogenic growth factors VEGF, FGF, PDGF produced by tumor cells can be inhibited by specific antibodies or antisense RNA or using gene specific SiRNA.

The targeted gene therapy can be achieved by using either viral vectors or nanoparticles carrying ligand RGD to endothelial cell surface specific receptors αvβ5, αvβ3 to target antiangiogenic genes to tumor vasculature. The inhibitors can also selectively express in EC using endothelial cell specific promoters like endothelin 1 EDT1.

Ad: adenovirus; AAV: adeno-associated virus; Retro: retrovirus; VEGF: vascular endothelial growth factor; FGF: fibroblast growth factor; PDGF: platelet derived growth factor; TNF: tumor necrosis factor; RGD: Arginine-glycine-aspartic acid; SiRNA: small interfering RNA.

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A recent study 7 has identified that the expression of a number of genes is increased in the tumor endothelium when compared with the nontumor endothelial cells.

Thus, because the tumor endothelium appears to have a number of altered features, the best candidate antiangiogenic molecules for cancer therapy should be directed at differences between the tumor vasculature and normal vasculature to increase specificity and efficacy.

However, many of the current antiangiogenic candidates also regulate similar activities, not only in tumor endothelium but also in normal endothelium and even in nontransformed cell types. The effectiveness of angiogenesis inhibitors in the treatment of various cancers remains to be determined.

Despite the possibility of tumor endothelium being heterogeneous and tumor type specific, antiangiogenesis inhibitors have been used in some 22 clinical trials to date. However, a major problem has been the availability amount and purity and the stability of some of these molecules.

Although there have been promising results in animal studies, the human trials for these protein-based inhibitors have been less successful 8.

The questions of how best to deliver the inhibitor, maintain its stability and activity, and target it to the tumor vasculature have not been answered.

Gene therapy represents an interesting alternative for the effective delivery of antiangiogenic therapy, and a number of studies 9 — 16 have demonstrated that a gene therapy-based antiangiogenesis approach is an effective means of reducing tumor growth in animal models.

Advantages of gene therapy over the direct administration of the inhibitors include the localized delivery and sustained expression of the antiangiogenic molecules, the ability to inhibit multiple angiogenic pathways with the delivery of more than one transgene, the generation of properly folded inhibitor molecules, and the potential for decreased cost 8 , Endostatin, a kd fragment of collagen XVIII with demonstrated antiangiogenic activity, is being tested in phase I clinical trials as a protein infusion for various cancers.

The gene for this protein is an ideal gene therapy candidate because the recombinant protein is difficult to produce and appears to be safe when delivered to the patient for an extended time. Endostatin has been tested in murine tumor models, with varying success, by gene therapy delivery vectors, including adenoviral vectors, adeno-associated viral vectors, in vitro transfections, polymerized plasmids, and DNA cationic liposomes 9 — 13 , In this issue of the Journal, Feldman et al.

They found that endostatin gene transfer led to the expression of functional endostatin and inhibited tumor growth. This study highlights a number of important points to consider in antiangiogenesis gene therapy.

First, there is no correlation between efficacy and the level of endostatin in the serum. For example, in the study by Feldman et al. One possibility for this latter observation is that effective inhibition of endothelial activity is dependent on both the ability to deliver endostatin to the right location and the response of the endothelial cells within the local tumor microenvironment.

Second, a lack of understanding of the mechanism s of action of endostatin has greatly hindered current efforts to improve its efficacy in vivo. In this respect, two recent studies 17 , 18 provide compelling data that a primary action of endostatin is the disruption of appropriate interactions between cell surface receptors and extracellular matrix proteins.

Currently, with the mechanism of action of a large number of putative antiangiogenic molecules unclear, any efforts to translate these research findings to clinical therapies may be premature.

Finally, the study by Feldman et al. However, retroviral vectors only transduce dividing cells, which may limit their in vivo use. Such limitations highlight the continued need for improved gene therapy vectors to overcome the current problems associated with various gene therapy vectors, including immune response to the vector, transduction efficiency, delivery of the gene to the target tissue, and controlled gene expression by the vector.

The general area of angiogenesis modulation is an exciting one, and gene therapy directed to inhibit angiogenesis can benefit from the more advanced gene therapy efforts to promote neovascularization 8. The early indications of success suggest that modulation of angiogenesis via gene therapy is reasonably safe and effective and offers considerable insight into the criteria for successful antiangiogenesis therapy.

First, effective therapeutic angiogenesis is based on the considerable knowledge of how VEGF and FGF work. Second, it is likely that multiple factors will be necessary to generate vessels that are stable over time.

Third, it remains unclear what is the best vector system to deliver optimal therapy. For researchers to realize the considerable advantages of antiangiogenesis gene therapy, it will be necessary to develop better vector platforms.

Additional challenges for antiangiogenesis therapy are to understand how angiogenesis inhibitors function, how tumor vessels differ from normal blood vessels, and how to target tumor vessels with appropriate combination therapies.

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Chitosan is another carrier derived from chitin. It is less cytotoxic and is biodegradable and metabolized easily by the kidneys.

In mice models of breast cancer, chitosan nanoparticles containing anti-Rho small interfering RNA siRNA showed tumour anti-angiogenesis [ 56 ]. The binding of αvβ3 integrin to chitosan nanoparticles is an important development.

The receptor for αvβ3 integrin is widely expressed in tumours and has shown potentials in ovarian cancer models. Encapsulation of paclitaxel with chitosan nanoparticles has shown efficacy in breast cancer [ 57 ]. There is now interest in the use of mesenchymal stem cells MSCs to deliver nanoparticles.

Hypoxic conditioning of such MSCs used as cell-based therapy can be used for aggressive tumours like glioblastoma multiforme since MSCs can traffic across the blood-brain barrier [ 53 ]. Blocking tumour stem cells via anti-angiogenic therapies is another theoretical approach since the tumour stem cell sub-population in some tumours like breast cancers may be more adept at promoting angiogenesis than their non-stem cell counterparts.

The different delivery methods for nanoparticles are compared in Table 2. Anti-angiogenic therapy in cancers has enormous potentials using VEGF signaling pathways. Cardiovascular toxicity and off-target effects of anti-angiogenic drugs are impediments to their long-term use in those at high cardiovascular risk.

Continued research into effective nanoparticle-based delivery methods is an exciting and developing field in cancer therapeutics. Understanding of the molecular and cellular mechanisms of tumour angiogenesis will facilitate the development of newer effective anti-angiogenic molecules.

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Oguntade, A. et al. Anti-angiogenesis in cancer therapeutics: the magic bullet. J Egypt Natl Canc Inst 33 , 15 Download citation. Received : 18 November Accepted : 08 June Published : 02 July Anyone you share the following link with will be able to read this content:.

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Search all SpringerOpen articles Search. Download PDF. Narrative Review Open access Published: 02 July Anti-angiogenesis in cancer therapeutics: the magic bullet Ayodipupo S.

Oguntade ORCID: orcid. Abstract Background Angiogenesis is the formation of new vascular networks from preexisting ones through the migration and proliferation of differentiated endothelial cells. Main body of the abstract MEDLINE and EMBASE databases were searched for publications on antiangiogenic therapy in cancer therapeutics from to Short conclusion Clinical surveillance is important for the early detection of tumour resistance and treatment failure using reliable biomarkers.

Background Cancers still account for significant morbidity and mortality globally despite remarkable advances in the management of cancers [ 1 ]. Main text We searched MEDLINE and EMBASE for publications on anti-angiogenesis in cancer from to as part of a larger project on anti-angiogenesis and cancer therapeutics.

Anti-angiogenics in cancers Several preclinical and clinical studies in cancer research have targeted different steps of the angiogenic pathway. Table 1 Selected VEGF-targeted anti-angiogenics and their therapeutic indications Full size table.

Clinical approach to cardiovascular toxicity of antiangiogenic therapy. Full size image. Table 2 Different delivery methods for nanoparticles Full size table. Conclusion Anti-angiogenic therapy in cancers has enormous potentials using VEGF signaling pathways.

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You can also search for this author in PubMed Google Scholar. Reprints and permissions. Malecki, M. Angiogenic and antiangiogenic gene therapy. Gene Ther 12 Suppl 1 , S—S Download citation. Published : 18 October Issue Date : 01 October Anyone you share the following link with will be able to read this content:.

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Skip to main content Thank you for visiting nature. nature gene therapy conference paper article. Abstract Gene therapy is thought to be a promising method for the treatment of various diseases.

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