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Adeno-associated viruses (AAVs) are currently the most popular vector platform technology for ocular gene therapy. While transduction efficiency and tropism of intravitreally administered AAV has been fairly well established in various retinal conditions, its transduction pattern in diabetic retinas has not previously been characterized. Here, we describe the transduction efficiencies of four different AAV serotypes, AAV2, 5, 8, and 9, in streptozotocin (STZ)-induced diabetic mouse retinas after intravitreal injections, which differed according to the duration of diabetic induction. STZ was intraperitoneally injected into C57/B6 diabetic mice subjected to unilateral intravitreal injection of AAV2, AAV5, AAV8, and AAV9 packaged with EGFP. Significantly enhanced AAV2 and AAV9 transduction was observed in 2-month-old diabetic mouse retinas compared to the 2-week-old diabetic mouse retinas and nondiabetic, vector uninjected or injected retinas. Intravitreal injection of AAV5 or AAV8 serotype in 2-month- and 2-week-old diabetic mouse retinas did not show any significant vector transduction enhancement compared to the nondiabetic control retinas. The tropism of AAV2 and AAV9 in diabetic mouse retinas differed. AAV2 was transduced into various retinal cells, including Müller cells, microglia, retinal ganglion cells (RGCs), bipolar cells, horizontal cells, and amacrine cells, whereas AAV9 was effectively transduced only into RGC and horizontal cells. The expression levels of receptors and co-receptors for AAV2 and AAV9 were significantly increased in 2-month-old diabetic mouse retinas. The results of our study demonstrated that AAV2 and AAV9 may be the vector of choice in treating diabetic retinopathy (DR) with gene therapy, and DR-related retinal changes may improve AAV vector transduction efficiency. Keywords: adeno-associated virus, diabetes mellitus, diabetic retinopathy, gene therapy

Choroidal neovascularization (CNV) is the defining characteristic of the wet subtype of age-related macular degeneration (AMD), which is a rapidly growing global health problem. Previously, we had demonstrated the therapeutic potential of gene therapy against CNV using short hairpin RNA (shRNA) delivered via recombinant adeno-associated virus (rAAV), which abrogates mammalian-to-mechanistic (mTOR) activity in a novel manner by simultaneously inhibiting both mTOR complexes. Both the target and use of gene therapy represent a novel treatment modality against AMD. Here, the xenogeneic GFP gene used as a reporter in previous studies was removed from the virus vector to further develop the therapeutic for clinical trials. Instead, a stuffer DNA derived from the 3′ UTR of the human UBE3A gene was used to ensure optimal viral genome size for efficient rAAV assembly. The virus vector containing the stuffer DNA, rAAV2-shmTOR-SD, positively compares to one encoding the shRNA and a GFP expression cassette in terms of reducing CNV in a laser-induced mouse model, as determined by fundus fluorescein angiography. These results were confirmed via immunohistochemistry using anti-CD31, while a TUNEL assay showed that rAAV2-shmTOR-SD possesses anti-apoptotic properties as well. The qualities exhibited by rAAV2-shmTOR-SD demonstrate its potential as a human gene therapeutic for the treatment of wet AMD.

Adeno-associated virus (AAV) vector is a promising platform technology for ocular gene therapy. Recently clinical successes to treat choroidal neovascularization (CNV) in wet type age-related macular degeneration have been reported. However, because pathologic conditions of the retina may alter the tropism of viral vectors, it is necessary to evaluate the transduction efficiency of different serotypes of AAV vectors in the retinas with CNVs. Here, we show the patterns and efficacy of transduction of AAV2, -5, and -8 vectors in a laser-induced CNV mouse model. C57BL/6J mice were subjected to unilateral laser photocoagulation on the right eye to induce CNV 5 days prior to intravitreal injection of AAV2, -5, and -8 capsids expressing EGFP. Transduction was increased around CNV lesions for all AAV capsid types, and AAV2 resulted in the highest transduction efficiency. In the absence of CNV, the AAV2 vector transduced ganglion and inner nuclear layer (INL) cells, and AAV5 and AAV8 transduced only a small proportion of cells in the retinal ganglion cell layer. CNV increased AAV2 vector expression throughout the retina and in and around CNVs; the transduced cells included retinal ganglion cells, Müller cells, cells from the INL and outer nuclear layer (ONL), photoreceptors, and retinal pigment epithelium (RPE) cells. Inflammatory cells and endothelial cells in CNVs were also transduced by AAV2. AAV5 and AAV8 were transduced in retinal ganglion, Müller, INL, ONL, and RPE cells in a localized pattern, and only endothelial cells at the surface of CNV lesions showed EGFP expression. Taken together, CNV formation resulted in enhanced transduction of AAV2, -5, and -8, and AAV2 exhibited the highest transduction efficiency in cells in CNV lesions. Keywords: adeno-associated virus, choroidal neovascularization, age-related macular generation, gene therapy

2 Division of Radiation Cancer, Korea Institute of Radiological and Medical Sciences (KIRAMS), Seoul 139-706, Korea - We have recently re- ported the PRC1 promoter as a promoter candidate to control expression of transcriptionally targeted genes for breast cancer gene therapy. We tested whether the PRC 1 promoter could be also applied for the lung cancer gene therapy. In the transient transfection assay with naked plasmids containing the luciferase fused to the PRC1 promoter, the promoter showed little activity in the normal lung cell line, MRC5. However, in the lung cancer A549 cells, PRC1 showed approximately 30-fold activa- tion which was similar to the survivin promoter, the gene whose promoter has been already re- ported as a candidate for the gene therapy of lung cancer. In viral systems, the PRC1 promoter showed approximately 75% and 66% of transcriptional activity compared to the CMV promoter in the adeno-associated virus (AAV) and the adenovirus (AV) systems, respectively. However, the PRC1 promoter in either AAV or AV showed approximately 20% activity compared to the CMV promoter in the normal lung cells. In addition, human lung tumor xenograft mice showed that the PRC1 promoter activity was as strong as the CMV activity in vivo. Taken together, these results sug- gested that PRC1 might be a potential promoter candidate for transcriptionally targeted lung cancer gene therapy.

To develop a novel therapeutic angiogenesis for the treatment of cardiovascular diseases, angiogenin (ANG1) was examined as a potential therapeutic gene. An adeno-associated virus (AAV)-mediated gene delivery system was used to measure the therapeutic efficacy of ANG1. Using a triple co-transfection technique, rAAV-ANG1-GFP, rAAV- VEGF-GFP and rAAV-GFP vectors were produced, which were then used to infect human umbilical vein endothelial cells (HUVECs) in order to evaluate in vitro angiogenic activities. Their protein expressions, tagged with green fluorescent protein (GFP), were monitored by confocal microscopy. The functional activities were measured using wound- healing HUVEC migration assays. The number of migrated cells stimulated by both the expressed ANG1 and the VEGF in rAAV-infected HUVECs increased almost twice the number observed in the expressed GFP control. In vivo angiogenic activities of the expressed ANG1 or VEGF were determined using mouse angiogenesis assays. The angiogenic activities of ANG1 or VEGF expressed in the injected mice were increased by 1.36 and 2.16 times, respectively, compared to those of the expressed GFP control. These results demonstrate that the expressed ANG1 derived from rAAV infection has in vitro and in vivo angiogenic activities and suggest that the rAAV-ANG1 vector is a potential strategy for therapeutic angiogenesis.

Mutations in human DRA cause congenital chloride diarrhea, thereby raising the possibility that it functions as a Cl(-)/HCO(3)(-) exchanger. To test this hypothesis we cloned a cDNA encoding mouse DRA (mDRA) and analyzed its activity in cultured mammalian cells. When expressed in HEK 293 cells, mDRA conferred Na(+)-independent, electroneutral Cl(-)/CHO(3)(-) exchange activity. Removal of extracellular Cl(-) from medium containing HCO(3)(-) caused a rapid intracellular alkalinization, whereas the intracellular pH increase following Cl(-) removal from HCO(3)(-)-free medium was reduced greater than 7-fold. The intracellular alkalinization in Cl(-)-free, HCO(3)(-)-containing medium was unaffected by removal of extracellular Na(+) or by depolarization of the membrane by addition of 75 mM K(+) to the medium. Like human DRA mRNA, mDRA transcripts were expressed at high levels in cecum and colon and at lower levels in small intestine. The expression of mDRA mRNA was modestly up-regulated in the colon of mice lacking the NHE3 Na(+)/H(+) exchanger. These results show that DRA is a Cl(-)/HCO(3)(-) exchanger and suggest that it normally acts in concert with NHE3 to absorb NaCl and that in NHE3-deficient mice its activity is coupled with those of the sharply up-regulated colonic H(+),K(+)-ATPase and epithelial Na(+) channel to mediate electrolyte and fluid absorption.

We report here the cloning, primary structure, heterologous expression, tissue distribution, and localization of a cDNA encoding rat NHE5, a fifth member of the mammalian plasma membrane Na+/H+ exchanger (NHE) gene family. The full-length open reading frame as well as 34 nucleotides of 5′-untranslated and 1443 nucleotides of 3′-untranslated sequences were obtained using a polymerase chain reaction strategy involving reverse transcription-polymerase chain reaction and 5′/3′-rapid amplification of cDNA ends. The NHE5 cDNA encodes a protein of 898 amino acids with a calculated M r of 99,044 and is predicted to contain 11–13 transmembrane domains. An amino acid comparison of the coding region of rat NHE5 reveals 95% identity with human NHE5. Northern hybridization analysis showed that high level expression of NHE5 mRNA is restricted to brain. Transfection of the coding region of rat NHE5 into NHE-deficient PS120 cells resulted in Na+/H+ exchange activity that was relatively insensitive to the amiloride analogue, 5-(N-ethyl-N-isopropyl) amiloride, with a half-maximal inhibitory concentration (IC50) of 1.53 ± 0.25 μm. In situ hybridization of rat brain sections revealed significant NHE5 mRNA levels in the dentate gyrus with lower levels observed in the hippocampus and cerebral cortex. These results suggest a specialized role for this fifth NHE isoform in neuronal tissues.

Rat parotid acinar cells express Cl- currents that are activated in a time-dependent manner by hyperpolarized potentials. ClC-2, a member of the ClC gene family, codes for a voltage-gated, inward rectifying anion channel when expressed in Xenopus oocytes. In the present study, we found that cDNA derived from individual parotid acinar cells contained sequence identical to that reported for ClC-2 in rat brain and heart. A polyclonal antibody generated against the N-terminal cytoplasmic domain of ClC-2 recognized an approximately 100 kD protein on western blots of both brain and parotid gland. ClC-2 expressed in oocytes has different kinetics from the currents found in parotid acinar cells. Since the ClC-2 channel was cloned from and its transcripts are expressed in mammalian tissue, we compared the channel properties of acinar cells to a mammalian expression system. We expressed ClC-2 channels in human embryonic kidney cells, HEK 293, using recombinant ClC-2 DNA and ClC-2 DNA fused with DNA coding for jellyfish green fluorescent protein (GFP). Confocal microscopy revealed that the expressed ClC-2-GFP chimera protein localized to the plasma membrane. Whole cell Cl- currents from HEK 293 cells expressing ClC-2-GFP were similar, if not identical, to the Cl- currents recorded from cells transfected with ClC-2 cDNA (no GFP). The voltage-dependence and kinetics of ClC-2 channels expressed in HEK 293 cells were quite similar to those in acinar cells. Channels in parotid acinar and HEK 293 cells activated at more positive membrane potentials and with a faster time course than the channels expressed in Xenopus oocytes. In summary, we found that ClC-2 message and protein are expressed in salivary cells and that the properties of voltage-activated, inward rectifying Cl- channels in acinar cells are similar to those generated by the ClC-2-GFP construct expressed in HEK 293 cells. The properties of the ClC-2 anion channel seem to be dependent on the type of cell background in which it is expressed.

Insulin induction of acetyl-CoA carboxylase (ACC) and differentiation of 30A5 preadipocytes into adipocytes requires a brief exposure of the cells to cAMP. Using the techniques of DNase I footprinting, DNA band shift, and analysis of the point and deletion mutations, a region from -113 to -95 has been identified as the site through which cAMP sensitizes the cell for the response to insulin. One sequence-specific DNA-protein complex, b3, is formed in the DNA-mobility shift assay when nuclear extract from 30A5 cells is mixed with the oligonucleotide representing this region. Purified human AP-2 also generates the complex corresponding to b3 with the same ACC PII probe or with the AP-2 consensus sequence probe from SV40 promoter. Substitution of A for G in the sequence GGGGCTGGG abolishes the formation of b3 sequence-specific complex. Stably transfected 30A5 cells with the same mutations in the plasmid no longer respond to insulin in spite of their exposure to cAMP. These results establish that the 21 base pair region in ACC promoter II and the binding of AP-2 protein to this sequence are required for cAMP action. cAMP-dependent protein kinase phosphorylates AP-2 both in vitro and in vivo and the phosphorylation of AP-2 does not affect its binding activity.

Tumour necrosis factor (TNF) inhibits the accumulation of acetyl CoA carboxylase (ACC) mRNA by decreasing the rate of ACC gene transcription. The ACC mRNA species found in 30A5 cells are generated from promoter II and TNF inhibits the accumulation of class 2 type mRNAs. By using 5' deletion mutants of promoter II fused to the bacterial chloramphenicol acetyltransferase (CAT) gene, the DNA mobility shift assay and the DNase I footprinting assay, the authors have identified the 30 bp from −389 to −359 as the TNF responsive element in promoter II. TNF treatment causes a decrease in the binding activity of nuclear protein(s) specific to the TNF responsive element. When the fragment containing the TNF responsive element was incorporated into the thymidine kinase promoter, the chimeric gene exhibited TNF induced inhibition of expression.

30A5 preadipocytes, derived from 10T1/2 mouse fibroblasts, can be induced to differentiate into adipocytes by hormone treatment. In this paper, we introduce a modified procedure to induce differentiation of 30A5 cells by pretreatment with cAMP for a brief period or by a "nutrition deprivation" pretreatment, followed by incubation in medium containing insulin. These procedures accelerate the differentiation of the preadipocytes, so that the cells are fully differentiated within 4 days instead of the 7-8 days normally required. This differentiation is accompanied by the early induction of acetyl-CoA carboxylase (ACC). ACC catalyzes the rate-limiting step in the biogenesis of long chain fatty acids. To analyze the relationship between cAMP and insulin action in the induction of ACC and cell differentiation, we identified the DNA sequences in promoter II of the ACC gene necessary for the action of insulin and cAMP. Chimeric genes between different fragments of the ACC promoter and the promoterless chloramphenicol transacetylase (CAT) gene were constructed, and stable clones containing these chimeric genes were obtained. By analyzing the CAT activities in these stable clones, we established that insulin action in inducing ACC and cell differentiation requires prior treatment of cells with cAMP and the presence of specific DNA regions in the ACC promoter for cAMP action. Stable clones containing a chimeric gene which consists of DNA sequences in promoter II that are required for insulin action, thymidine kinase promoter, and the CAT gene did not respond to insulin. However, when the DNA sequences required for cAMP action were placed in this chimeric gene, it responded to insulin upon prior treatment of 30A5 cells with cAMP. Thus, cAMP and insulin, whose physiological actions generally appear to be antagonistic, are synergistically interacting in the induction of ACC and the differentiation of 30A5 cells.

Herpesvirus saimiri (HVS), a member of the gamma-herpesvirus family, encodes an oncoprotein called Saimiri Transforming Protein (STP) which is required for lymphoma induction in non-human primates. However, a detailed mechanism of STP-A11-induced oncogenesis has not been revealed yet. We first report that STP-A11 oncoprotein interacts with TNF-alpha receptor-associated factor (TRAF) 6 in vivo and in vitro. Mutagenesis analysis of the TRAF6-binding motif (10)PQENDE(15) in STP-A11 reveals that Glu (E)(12) residue is critical for binding to TRAF6 and NF-kappaB activation. Interestingly, co-expression of E12A mutant, lack of TRAF6 binding, with cellular Src (Src) results in decreased transcriptional activity of Stat3 and AP-1, a novel target of STP-A11 compared to that of wild type. Furthermore, the presence of STP-A11 enhances the association of TRAF6 with Src and induces the translocation of both TRAF6 and Src to a nonionic detergent-insoluble fraction. Taken together, these studies suggest that STP-A11 oncoprotein up-regulates both NF-kappaB and AP-1 transcription activity through TRAF6, which would ultimately contribute cellular transformation.

Although the human telomerase reverse transcriptase (hTERT) promoter can regulate cancer-specific genes, it is generally too weak to be effective. We therefore attempted to improve the potency of synthetic hTERT promoters by fusing the core element (E) of the hTERT promoter (H) and the tripartite leader sequence (T) from human adenovirus 5 in a combinatorial manner. To determine the potential as cancer-specific promoters, we measured luciferase activity driven by the chimeric hTERT promoters in human cancer cells. Among various constructs, the E3-H-T promoter induced the strongest luciferase activity in all the tested cancer cells. SK-Hep1 and Hela cells experienced 1000- and 11-fold higher expression than the basic hTERT promoter, respectively. Relative to the SV40 universal promoter, the E3-H-T promoter led to higher levels of gene expression. Using EMSA, we found that the hTERT enhancer region was specifically bound to c-Myc and Sp1. Thus, the data suggest that the E3-H-T promoter with up-regulated cancer-specific gene expression could be useful in cancer gene therapy.

T-cell factor (TCF)-1 protein forms the transcriptional complex with β-catenin and regulates the expression of diverse target genes during early development and carcinogenesis. We have selected previously an RNA aptamer that binds to the DNA-binding domain of TCF-1 and have shown that it interfered with binding of TCF-1 to its specific DNA recognition sequences in vitro. As an approach to modulate the transcription by TCF/β-catenin complex in the cells, we have developed the RNA expression vector for stable expression of RNA aptamer inside of the mammalian cells. High level of RNA was expressed as an intramer in the fusion with the stable RNA transcript. The RNA intramer inhibited TCF/β-catenin transcription activity as shown by luciferase assay. It also modulated the expression of TCF/β-catenin target genes, such as cyclin D1 and matrix metalloproteinase-7, as predicted to be as an effective inhibitor of the TCF function. In addition, it efficiently reduced the growth rate and tumorigenic potential of HCT116 colon cancer cells. Such RNA intramer could lead to valuable gene therapeutics for TCF/β-catenin-mediated carcinogenesis. [Mol Cancer Ther 2006;5(9):2428–34]

Multiple Na(+)/H(+) exchangers (NHEs) are expressed in salivary gland cells; however, their functions in the secretion of saliva by acinar cells and the subsequent modification of the ionic composition of this fluid by the ducts are unclear. Mice with targeted disruptions of the Nhe1, Nhe2, and Nhe3 genes were used to study the in vivo functions of these exchangers in parotid glands. Immunohistochemistry indicated that NHE1 was localized to the basolateral and NHE2 to apical membranes of both acinar and duct cells, whereas NHE3 was restricted to the apical region of duct cells. Na(+)/H(+) exchange was reduced more than 95% in acinar cells and greater than 80% in duct cells of NHE1-deficient mice (Nhe1(-/-)). Salivation in response to pilocarpine stimulation was reduced significantly in both Nhe1(-/-) and Nhe2(-/-) mice, particularly during prolonged stimulation, whereas the loss of NHE3 had no effect on secretion. Expression of Na(+)/K(+)/2Cl(-) cotransporter mRNA increased dramatically in Nhe1(-/-) parotid glands but not in those of Nhe2(-/-) or Nhe3(-/-) mice, suggesting that compensation occurs for the loss of NHE1. The sodium content, chloride activity and osmolality of saliva in Nhe2(-/-) or Nhe3(-/-) mice were comparable with those of wild-type mice. In contrast, Nhe1(-/-) mice displayed impaired NaCl absorption. These results suggest that in parotid duct cells apical NHE2 and NHE3 do not play a major role in Na(+) absorption. These results also demonstrate that basolateral NHE1 and apical NHE2 modulate saliva secretion in vivo, especially during sustained stimulation when secretion depends less on Na(+)/K(+)/2Cl(-) cotransporter activity.

Several members of the Na+/H+exchanger gene family (NHE1, NHE2, NHE3, and NHE4) with unique functional properties have been cloned from rat epithelial tissues. The present study examined the molecular and pharmacological properties of Na+/H+exchange in rat parotid salivary gland cells. In acinar cells superfused with a physiological salt solution (145 mM Na+), Na+/H+exchanger activity was inhibited by low concentrations of the amiloride derivative ethylisopropyl amiloride (EIPA; IC50= 0.014 ± 0.005 μM), suggesting the expression of amiloride-sensitive isoforms NHE1 and/or NHE2. Semiquantitative RT-PCR confirmed that NHE1 transcripts are most abundant in this cell type. In contrast, the intermediate sensitivity of ductal cells to EIPA indicated that inhibitor-sensitive and -resistant Na+/H+exchanger isoforms are coexpressed. Ductal cells were about one order of magnitude more resistant to EIPA (IC50= 0.754 ± 0.104 μM) than cell lines expressing NHE1 or NHE2 (IC50= 0.076 ± 0.013 or 0.055 ± 0.015 μM, respectively). Conversely, ductal cells were nearly one order of magnitude more sensitive to EIPA than a cell line expressing the NHE3 isoform (IC50= 6.25 ± 1.89 μM). Semiquantitative RT-PCR demonstrated that both NHE1 and NHE3 transcripts are expressed in ducts. NHE1 was immunolocalized to the basolateral membranes of acinar and ductal cells, whereas NHE3 was exclusively seen in the apical membrane of ductal cells. Immunoblotting, immunolocalization, and semiquantitative RT-PCR experiments failed to detect NHE2 expression in either cell type. Taken together, our results demonstrate that NHE1 is the dominant functional Na+/H+exchanger in the plasma membrane of rat parotid acinar cells, whereas NHE1 and NHE3 act in concert to regulate the intracellular pH of ductal cells.

For cancer gene therapy, cancer-specific over- expression of a therapeutic gene is required to reduce side effects derived from expression of the gene in normal cells. To develop such an expression vector, we searched for genes over-expressed and/or specifically expressed in cancer cells using bioinformatics and have selected genes coding for protein regulator of cytokinesis 1 (PRC1) and ribonuclease reductase 2 (RRM2) as candidates. Their cancer-specific expressions were confirmed in both breast cancer cell lines and patient tissues. We compared each promoter's cancer-specific activity in the breast normal and cancer cell lines using the luciferase gene as a reporter and confirmed cancer-specific expression of both PRC1 and RRM2 promoters. To test activities of these promoters in viral vectors, the promoters were also cloned into an adeno-associated viral (AAV) vector containing green fluorescence protein (GFP) as the reporter. The GFP expression levels by these promoters were various depending on cell lines tested and, in MDA-MB-231 cells, GFP activities derived from the PRC1 and RRM2 promoters were as strong as that from the cytomegalovirus (CMV) promoter. Our result showed that a vector containing the PRC1 or RRM2 promoter could be used for breast cancer specific overexpression in gene therapy.

Over the past several years, viral and non-viral gene delivery systems have been intensively developed to establish an ideal delivery vector for cancer gene therapy. Among the large variety of virus vectors currently being developed, the vector based on recombinant AAV (rAAV) is one of those that are closest to the ideal vector due to the following features; the lack of pathogenicity and toxicity, ability to infect dividing and non-dividing cells of various tissue origins, a very limited host immune response and long-term persistent expression. However, there is concern that two major obstacles of AAV serotype 2 (AAV2), the relative paucity of AAV receptors on certain cell types and the presence of pre-existing immunity to AAV2 in humans might limit its clinical applications in humans. To overcome these limitations, we first investigated in vitro transduction efficiencies for 7 different AAV serotypes (AAV1 |[ndash]| AAV6, AAV8) in 12 human tumor cell lines (MKN74, AGS, NCI-H460, HT1080, HepG2, SK-Hep1, HCT-116, 5637, UMUC3, HeLa, DU145 and MCF7). We employed |[ldquo]|pseudotyped|[rdquo]| AAV vector systems, in which rAAV2 vector genomes containing the CMV promoter/ enhancer, the green fluorescent protein (GFP) cDNA and SV40 poly (A) signal, flanked by AAV2 ITR sequences are cross-packaged into the capsid proteins of the other AAV serotypes (several plasmids are kindly provided by Dr. Katherine A. High, University of Pennsylvania Medical Center). As we reported previously, the efficiency of rAAV2 infection into most of the tested cancer cell lines was significantly high (>90%), whereas the transduction efficiencies for other 5 different rAAV serotypes were varied and greatly low. On the other hand the efficiency of rAAV5 transduction in various tumor cell lines were comparably high, mostly similar to that of rAAV2. Using the xenograft nude mice implanted with either NCI-H460 or HCT-116 cancer cells, we also studied in vivo transduction efficiency for several pseudotyped rAAV vectors, which were shown to have a host range different from rAAV2. Taken altogether our results suggest that the pseudotyped rAAV5 vector can be developed as one of the most efficient gene delivery systems for cancer gene therapy to overcome the anti-AAV2 immune response. (This study was supported by the grant from the Ministry of Science and Technology (M10534040005-05N3404-00511), Seoul, and the Chung-Buk Pioneering Bioindustry R&D Grant, Chung-Buk, Korea.)

Gene therapy has offered potential promises for treatment of cancers, as a variety of genes controlling molecular processes can be introduced by gene transfer, which can arrest angiogenesis, tumor growth, invasion and/or metastasis. The establishment of an angiogenic requirement for tumor growth led to develop anti-angiogenic therapies, ranging from inhibition of expression of angiogenic molecules, through overexpression of antiangiogenic factors. Although adenovirus-based vector has been commonly used for this strategy due to advantages of gene therapy over purified antiangiogenic factors, gene expression mediated by adenoviral vector is greatly limited by an effective host immune response and also secondary to the episomal nature of the vector. However, the advantages of rAAV over other vectors are multiple, including a non-pathogenic vector with a very limited host immune response and long-term persistent expression of AAV transgenes. Here we investigated the potential anti-angiogenic cancer gene therapy of AAV-mediated gene delivery system containing antisense VEGF-A (rAAV-AShVEGF-A). The efficiency of rAAV transduction of T84, LCSC#1, MKN45, MKN74 and NUGC3 human cancer cell lines is significantly high (>90%). The expression of VEGF-A was greatly reduced (>60%) when T84 and LCSC#1 were treated with rAAV-AShVEGF-A. Moreover, the growth of tumor implanted in nude mice were highly inhibited (>40%) by injection of rAAV-AShVEGF-A. We also performed the preclinical safety evaluations of repeated dose toxicity, immunotoxicity, genotoxicity and reproductive performance toxicity on rAAV vectors. No results of preclinical tests that we carried out in this study revealed any adverse effects as a gene delivery system.

Top of pageAbstract A promising therapeutic angiogenesis using gene transfer of angiogenic regulators has been extensively investigated and developed for the treatment of various diseases including cardiovascular diseases such as coronary and peripheral artery disease, ischemic disease, restenosis, vein-graft failure, as well as ulcers. Many clinical trials and animal studies have shown that administration of angiogenic factors can increase permeability and nutrient perfusion to induce the formation of supplemental collateral blood vessel. In those studies, the best angiogenic factors are vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). However, other angiogenic factors showing better angiogenic activity need to be screened. Although angiogenin has been known that it interacts with endothelial and smooth muscle cells to induce a wide range of cellular responses including cell migration, invasion, proliferation, and neovascularization, a possibility of therapeutic approach using angiogenin remains unclear. Here we investigated a potential therapeutic angiogenesis using a naked DNA containing angiogenin cDNA. Angiogenin cDNA that contains either of whole open reading frame including signal peptide (SPANG1) or only mature peptide region (ANG1) was inserted into the linearized pEGFP-N1 vector (Clontech) to generate two different subclones, so called pSPANG1-EGFP and pANG1-EGFP. HEK293 or TSA-201 cells (a transformed HEK293 cell line that stably expresses the SV40 T-antigen) were used for transfection to express those of two different constructs. The angiogenin-GFP protein expression was monitored by a confocal microscopy and the expression pattern of angiogenin-GFP was examined using western blot analyses. Both of angiogenin-GFP proteins containing the signal peptide or not migrated to the same position of 43 kD. The angiogenin-GFP protein containing the signal peptide in the cell lysates began to be detected at 48 hr post-transfection, whereas the angiogenin-GFP protein of the mature peptide in the cell lysates was first detected at 24 hr after transfection. On the other hand, the secreted angiogenin-GFP from the cells expressing pSPANG1-EGFP was first detected at 72 hr post-transfection, in contrast, the secreted angiogenin-GFP from the cells expressing pANG1-EGFP was first measurable at 24 hr after transfection. The angiogenin-GFP expression from the cells transfected with both type of constructs was maintained to 96 hr post-transfection. The functional activity of the expressed angiogenin-GFP proteins was tested by a wound-healing assay. More studies using pCIneo-SPANG1 and pCIneo-ANG1 are currently under investigation to verify that GFP tagging does not interfere angiogenic activity of angiogenin. Animal studies using four different types of naked DNA containing angiogenin cDNA remain to be done. Taken together, these data suggest that the naked DNA containing angiogenin cDNA may be a potential therapeutic angiogenesis for the treatment of cardiovascular diseases.

Acetyl-CoA carboxylase [acetyl-CoA:carbondioxide ligase (ADP-forming), EC 6.4.1.2] is the rate-limiting enzyme in the biogenesis of long-chain fatty acids. We have previously characterized five acetyl-CoA carboxylase mRNA species that differ in their 5' untranslated regions but not in the coding region. We have now characterized the exon-intron structure of the genomic DNA that encodes the 5' untranslated region of the mRNA. Generation of different forms of the mRNA is the result of the selective use of two promoters and differential splicing of five different exons. These five exons contain a total of 645 nucleotides and they are scattered over a 50-kilobase-pair genomic DNA region that we have characterized.