A system for the propagation of adenoviral vectors with genetically modified receptor specificities.

The development of genetically modified adenovirus (Ad) vectors with specificity for a single cell type will require both the introduction of novel tropism determinants and the ablation of endogenous tropism. Consequently, it will not be possible to exploit the native cellular entry pathway in the propagation of these targeted Ad vectors. Based on the concept that Ad enters cells by a two-step process in which a primary receptor serves as a high affinity binding site for the Ad fiber knob, with subsequent internalization mediated by alpha v integrins, we designed two artificial primary receptors. The extracellular domain of one of these synthetic receptors was derived from a single-chain antibody (sFv) with specificity for Ad5 knob, while the second receptor consisted of an icosapeptide identified by biopanning a phage display library against Ad5 knob. Expression of either of these artificial virus-binding receptors in fiber receptor-negative cells possessing alpha v integrins conferred susceptibility to Ad infection. We then created a novel mechanism for cell binding by genetically modifying both the vector and the target cell. In this approach, six histidine (His) residues were incorporated at the C-terminal of the Ad fiber protein. The resultant Ad vector was able to infect nonpermissive cells displaying the cognate artificial receptor, containing an anti-His sFv. This strategy, comprising a genetically engineered Ad virion and a modified cell line, should be useful in the propagation of targeted Ad vectors that lack the ability to bind the native fiber receptor.

Advanced generation adenoviral vectors possess augmented gene transfer efficiency based upon coxsackie adenovirus receptor-independent cellular entry capacity.

Adenoviral (Ad) vectors have been widely used in the context of cancer gene therapy approaches. Their utility in these contexts, however, has frequently been limited by tumor cell resistance to Ad infection. The basis of this resistance has been defined recently as resulting from a deficiency of the primary adenovirus receptor, coxsackie adenovirus receptor. As a means to circumvent this limitation, a variety of tropism modification strategies have allowed coxsackie adenovirus receptor-independent gene delivery via the Ad vector. These advanced generation adenovirus vectors exhibit enhanced infectivity, which can allow direct therapeutic gain. Such vectors may allow improvements in efficacy in the context of ongoing human clinical gene therapy approaches for cancer.

Fiber knob modifications overcome low, heterogeneous expression of the coxsackievirus-adenovirus receptor that limits adenovirus gene transfer and oncolysis for human rhabdomyosarcoma cells.

Exploiting the lytic life cycle of viruses has gained recent attention as an anticancer strategy (oncolysis). To explore the utility of adenovirus (Ad)-mediated oncolysis for rhabdomyosarcoma (RMS), we tested RMS cell lines for Ad gene transduction and infection. RMS cells were variably transduced by Ad. Compared with control cells, RMS cells were less sensitive or even resistant to oncolysis by wild-type virus. RMS cells expressed the Ad internalization receptors, alpha(v) integrins, but had low or undetectable expression of the major attachment receptor, coxsackievirus-Ad receptor (CAR). Mutant Ads with ablated CAR binding exhibited only 5-20% of transgene expression in RMS cells seen with a wild-type vector, suggesting that residual or heterogeneous CAR expression mediated the little transduction that was detectable. Immunohistochemical analysis of archived clinical specimens showed little detectable CAR expression in five embryonal and eight alveolar RMS tumors. Stable transduction of the cDNA for CAR enabled both efficient Ad gene transfer and oncolysis for otherwise resistant RMS cells, suggesting that poor CAR expression is the limiting feature. Gene transfer to RMS cells was increased >2 logs using Ads engineered with modified fiber knobs containing either an integrin-binding RGD peptide or a polylysine peptide in the exposed HI loop. The RGD modification enabled increased oncolysis for RMS cells by a conditionally replicative Ad, Ad delta24RGD, harboring a retinoblastoma-binding mutation in the E1A gene. Thus, the development of replication-competent vectors targeted to cell surface receptors other than CAR is critical to advance the use of Ad for treating RMS.

A conditionally replicative adenovirus with enhanced infectivity shows improved oncolytic potency.

The absence or the presence of low levels of the Coxsackievirus and adenovirus receptor (CAR) on several tumor types might limit the efficacy of recently proposed tumor-specific or conditionally replicative adenoviruses (CRAds). To address this issue, we used a genetic modification of the fiber knob in the context of an E1A-defective CRAd to allow CAR-independent target cell infection as a means to enhance oncolytic potency. Such infectivity-enhanced CRAd showed higher replication, more efficient infection, and lysis of tumor cells in vitro. Of note, the improved antitumor effect of the fiber-modified CRAd could be demonstrated in vivo. We conclude that the combination of genomic modification to achieve tumor-selective replication and capsid modification to enhance infectivity yields more potent oncolytic adenoviruses for use in cancer treatment.

Selective gene delivery to head and neck cancer cells via an integrin targeted adenoviral vector.

In vivo cancer gene therapy approaches for squamous cell carcinoma of the head and neck (SCCHN) based on adenoviral vector-mediated gene delivery have been limited by the suboptimal efficacy of gene transfer to tumor cells. We hypothesized that this issue was due to deficiency of the primary adenoviral receptor, the coxsackie-adenovirus receptor (CAR), on the tumor targets. Studies of CAR levels on SCCHN cell lines confirmed that their relative refractoriness to the adenoviral vector was based on this deficiency. To circumvent this deficiency, we applied an adenoviral vector targeted to a tumor cell marker characteristic of SCCHN. In this regard, integrins of the alpha2beta1 and alpha3beta1 class are frequently overexpressed in SCCHN. Furthermore, these integrins recognize the RGD peptide motif. On this basis, we applied an adenoviral vector genetically modified to contain such a peptide within the HI loop of the fiber protein as a means to alter viral tropism. Studies confirmed that the CAR-independent gene delivery achieved via this strategy allowed enhanced gene transfer efficiencies to SCCHN tumor cells. Importantly, this strategy could achieve preferential augmentation of gene transfer in tumor cells compared with normal cells. The ability to achieve enhanced and specific gene transfer to tumor cells via adenoviral vectors has important implications for gene therapy strategies for SCCHN and for other neoplasms in general.

Adenoviral-mediated delivery of herpes simplex virus thymidine kinase results in tumor reduction and prolonged survival in a SCID mouse model of human ovarian carcinoma.

The herpes simplex virus thymidine kinase gene is the most widely utilized toxin for selective killing of carcinoma cells. Expression of the viral thymidine kinase gene renders cells sensitive to the toxic effects of nucleoside analogs such as ganciclovir. An advantage of this system is the "bystander effect" whereby thymidine kinase transduced tumor cells elicit a toxic effect on surrounding nontransduced tumor cells. Ovarian carcinoma appears to be an ideal candidate for gene therapy as the majority of women present with advanced stage disease, have poor prognosis for long-term survival and have the disease confined within the peritoneal cavity. Therefore the utility of an adenoviral vector to elicit an in vitro bystander effect in ovarian carcinoma cells and the therapeutic efficacy of such a system in vivo was undertaken. Immunocompetent animals were utilized to determine the maximum dose of adenovirus that could be administered without any undesirable side effects and that preimmunization had no effects on subsequent challenge. SCID mice were orthotopically transplanted with human ovarian carcinoma cells and, after establishment of tumor, given a recombinant adenovirus expressing either the herpes simplex virus thymidine kinase or the Escherichia coli beta-galactosidase gene. Half the animals from each viral group were treated with either a ganciclovir regiment (50 mg/kg daily for 14 days) or an equal volume of serum-free media. A subset of mice were killed following drug treatment and analyzed for tumor reduction. The remaining animals were followed daily for survival. The animals treated with the recombinant adenovirus expressing the herpes simplex virus thymidine kinase gene and ganciclovir had significant reduction in overall tumor burden and demonstrated statistically significant prolongation in overall survival.

Using a tropism-modified adenoviral vector to circumvent inhibitory factors in ascites fluid.

Peritoneal compartmentalization of advanced stage ovarian cancer provides a rational scenario for gene therapy strategies. Several groups are exploring intraperitoneal administration of adenoviral (Ad) vectors for this purpose. We examined in vitro gene transfer in the presence of ascites fluid from ovarian cancer patients and observed significant inhibition of Ad-mediated gene transfer. The inhibitory activity was not identified as either complement or cellular factors, but depletion of IgG from ascites removed the inhibitory activity, implicating neutralizing anti-Ad antibodies. A wide range of preexisting anti-Ad antibody titers in patient ascites fluid was measured by ELISA. Western blot analysis demonstrated that the antibodies were directed primarily against the Ad fiber protein. To circumvent inhibition by neutralizing antibodies, a genetically modified adenoviral vector was tested. The Ad5Luc.RGD vector has an Arg-Gly-Asp (RGD) peptide sequence inserted into the fiber knob domain and enters cells through a nonnative pathway. Compared with the conventional Ad5 vector, Ad5Luc.RGD directed efficient gene transfer to cell lines and primary ovarian cancer cells in the presence of ascites fluid containing high-titer neutralizing anti-Ad antibodies. These results suggest that such modified Ad vectors will be needed to achieve efficient gene transfer in the clinical setting.

Biochemical characterization of VspI methyltransferase.

The gene (vspIM) encoding VspI methyltransferase (MTase) has previously been cloned and sequenced, and shown to belong to the gamma class of m6-adenine MTases [Degtyarev et al., Nucleic Acids Res. 21 (1993) 2015]. Here it is shown that the MTase modifies the third adenine within the recognition sequence 5'-ATTAAT-3'.

The therapeutic efficacy of adenoviral vectors for cancer gene therapy is limited by a low level of primary adenovirus receptors on tumour cells.

Replication-defective adenoviral vectors are currently being employed as gene delivery vehicles for cancer gene therapy. To address the hypothesis that the therapeutic efficacy of adenoviral vectors is restricted by their inability to infect tumour cells expressing low levels of the primary cellular receptor for adenoviruses, the coxsackievirus and adenovirus receptor (CAR), we have employed a pair of ovarian cancer cell lines differing only in the expression of a primary receptor for Ad5. This novel system thus allowed the direct evaluation of the relationship between the efficacy of an adenoviral vector and the primary receptor levels of the host cancer cell, without the confounding influence of other variable cellular factors. We demonstrate that a deficiency of the primary cellular receptor on the tumour cells restricts the efficacy of adenoviral vectors in two distinct cancer gene therapy approaches, TP53 gene replacement therapy and herpes simplex virus thymidine kinase/ganciclovir suicide gene therapy. Moreover, we show that a deficiency of the primary receptor on the tumour cells limits the efficiency of adenovirus-mediated gene transfer in vivo. Since a number of studies have reported that primary cancer cells express only low levels of CAR, our results suggest that strategies to redirect adenoviruses to achieve CAR-independent infection will be necessary to realize the full potential of adenoviral vectors in the clinical setting.

Genetic modification of liver grafts with an adenoviral vector encoding the Bcl-2 gene improves organ preservation.

BACKGROUND: Liver function after transplantation is determined by the quality of the donor organ and the influences of preservation, flush, and reperfusion injury. In this regard, cell death (apoptosis) plays an important role in organ preservation and rejection. Therefore, we examined the possibility of genetic modification of the liver graft with a recombinant adenovirus vector encoding the Bcl-2 gene to reduce apoptosis during the preservation time. METHODS: Liver grafts from C57B1/6 mice were procured and preserved using standard techniques. A replication defective adenovirus vector (deltaE1) containing the human Bcl-2 gene (AdCMVhBcl-2) was developed in our laboratory. An adenovirus vector encoding an irrelevant gene (Escherichia coli beta-galactosidase) was used as a control. Each mouse received 1 x 10(9) plaque forming units administered i.v. 48 hr before the liver procurement. Analyses of liver enzyme activities were determined in the preservation solution. Apoptosis in liver biopsies was determined by DNA fragmentation with an in situ histochemical assay. RESULTS: Immunohistochemical analysis and RT-PCR confirmed the expression of hBcl-2 in the grafts. Grafts from livers expressing hBcl-2 showed significant reduction of the aspartame amino transferase (AST) and lactate dehydrogenase (LDH) release compared with grafts from the control groups. After rewarming, significant cytoprotection was also observed in grafts from animals treated with AdCMVhBcl-2. Histological analysis correlated with the hepatocellular injury determined with transaminases and LDH in the preservation solution. Significant reduction in the number of apoptotic cells was observed in grafts expressing hBcl-2. CONCLUSIONS: We have demonstrated a novel approach to reducing the preservation injury to liver grafts with the human Bcl-2 gene. This approach may allow a longer preservation time, potentially reduce the incidence of primary nonfunction, decrease the immunogenicity of the cold injured organ, and increase the safer use of "marginal" liver grafts.

Bacterial expression and characterization of a cDNA for human liver estrogen sulfotransferase.

A distinct human estrogen sulfotransferase (hEST-1) cDNA has been isolated from a human liver lambda Zap cDNA library using a PCR procedure. The enzymatically active protein has been expressed in two bacterial expression systems and the kinetic and immunologic properties of the enzyme have been characterized. The full-length cDNA for hEST-1 is 994 base pairs in length and encodes a 294 amino acid protein with a calculated molecular mass of 35,123 Da. Purified hEST-1 migrated with an apparent molecular mass of 35,000 Da during SDS-polyacrylamide gel electrophoresis. Immunoblot analysis of hEST-1 expressed in E. coli with a rabbit anti-hEST-1 antibody yields a band of approximately 35,000 Da. The anti-hEST-1 antibody also detects a single band in human liver and jejunum cytosol which migrates with the same molecular mass as expressed hEST-1. There was also no cross-reactivity of hEST-1 with rabbit anti-hP-PST or rabbit anti-hDHEA-ST antibodies upon immunoblot analysis. hEST-1 was expressed in bacteria and purified to homogeneity. Expressed hEST-1 activity has a significantly greater affinity for estrogen sulfation than that found for the other human STs which conjugate estrogens. hEST-1 maximally sulfates beta-estradiol and estrone at concentrations of 20 nM. hEST-1 also sulfates dehydroepiandrosterone, pregnenolone, ethinylestradiol, and 1-naphthol, at significantly higher concentrations; however, cortisol, testosterone and dopamine are not sulfated. The results presented in this paper describe the expression and characterization of a human EST distinct from other human STs which sulfate estrogens. The high affinity of hEST-1 for estrogens indicates that this ST may be important in both the metabolism of estrogens and in the regulation of their activities.

Isolation and expression of an isoform of rat estrogen sulfotransferase.

A new isoform of rat liver estrogen sulfotransferase (EST), rEST-6, which is distinct from the previously reported rat EST [Demyan et al., Molec. Endocrinol. 6 (1992) 589], has been cloned, expressed, purified and characterized. A PCR procedure using oligonucleotide primers synthesized to the 5'-nontranslated and 3'-nontranslated regions of the published rEST sequence was used to isolate rEST-6 cDNA. The cloned DNA is 1000 bp in length and encodes a protein of 295 amino acids with a calculated molecular mass of 35,300 Da. rEST-6 is selectively expressed in male rats, as confirmed by Northern blot and immunoblot analyses. Northern blot analysis of male and female rat liver RNA with the rEST-6 cDNA as a probe shows a band with male RNA but not with female RNA. Similarly, immunoblot analysis of male and female rat liver cytosols with an antibody to rat EST yields a strong immunoreactive band in rat liver cytosol from male rats but not from females. Subsequent to bacterial expression and purification of rEST-6, the enzyme was analyzed kinetically and shown to sulfate estrogens but not dehydroepiandrosterone, pregnenolone, cortisol or testosterone. Maximal sulfation activity towards both beta-estradiol and estrone occurred at a concentration of 1 microM with substrate inhibition at higher concentrations. These results indicate that multiple, closely related forms of EST are present in rat liver. Analysis of the activity and regulation of these different EST enzymes is important in understanding estrogen metabolism in rats.

[Study of the structure-function organization of the variola virus genome. IV. Sequencing and analysis of the nucleotide sequence of the right terminus of the India-1967 strain genome]. / Izuchenie strukturno-funktsional'noi organizatsii genoma virusa natural'noi ospy. IV. Sekvenirovanie i analiz posledovatel'nosti nukleotidov pravogo kontsa genoma shtamma Indiia-1967.

Sequencing and computer analysis of the variola major virus strain India-1967 (VAR-IND) genome segment (53,018 bp) from the right terminal region have been carried out. Fifty nine potential open reading frames (ORFs) of over 60 amino acid residues have been identified. Structure-function organization of VAR-IND DNA segment under study was compared with the previously reported sequences from the analogous genomic regions of vaccinia virus strains Copenhagen (VAC-COP) and Western Reserve (VAC-WR) and variola virus strain Harvey (VAR-HAR). Multiple distinctions in the genetic map of VAR-IND from VAC-COP and VAC-WR have been revealed along with the high similarity to the corresponding VAR-HAR segment. Possible functions of the predicted viral proteins and the effect of their differences on the features of orthopoxviruses are discussed.

[Chemico-enzymatic synthesis of genetic elements for expression of synthetic genes in Bacillus subtilis cells]. / Khimiko-fermentativnyi sintez geneticheskikh élementov dlia ékspressii iskusstvennykh genov v kletkakh Bacillus subtilis.

In order to obtain the recombinant Bacillus subtilis strain, a transcriptional-translational control unit of the alpha-amylase gene of B. amyloliquefaciens was synthesized. The oligodeoxyribonucleotides were prepared by the modified triester method in solution and by the solid-phase approach. Then these oligonucleotides were joined by DNA ligase into two fragments which were cloned in the phage M13mp9 DNA and the plasmid pBR327. A plasmid harboring the site regulating the transcription of the alpha-amylase gene may be employed as vector for cloning the promoter-containing fragments in E. coli cells.

[Expression in Escherichia coli cells of the nucleotide sequence of DNA coding for the bovine lipotropic hormone]. / Ekspressiia v kletkakh Escherichia coli nukleotidnoi posledovatel'nosti DNK, kodiruiushchei lipotropnyi gormon byka.

A cDNA fragment of bovine proopiomelanocortin coding for beta-lipotropic hormone was joined with a promoter and ribosome binding site of B. amyloliquefaciens and cloned in E. coli in pBR 327 plasmid. The level of beta-lipotropin synthesis in bacterial cells transformed by the obtained plasmid was estimated immunochemically. The level of beta-lipotropin production was shown to be 5 mg per liter of bacterial culture.

Oncolytic adenovirus retargeted to Delta-EGFR induces selective antiglioma activity.

The fact that glioblastomas, which are one of the most devastating cancers, frequently express the Delta-EGFR (epithelial growth factor receptor) also called mutant variant III of EGFR (EGFRvIII) suggests that this cancer cell-specific receptor might serve as an ideal target for cancer therapy. To assess its potential as such a target, we constructed an oncolytic adenovirus with Retargeted Infectivity Via EGFR (Delta-24-RIVER) on the backbone of Delta-24. This new oncolytic adenovirus targets, as Delta-24 does, the disrupted Rb pathway in cancer cells; in addition, this adenovirus has also been retargeted through the abrogation of CAR binding (Y477A mutation in adenoviral fiber protein) and insertion of an EGFRvIII-specific binding peptide in the HI loop of the fiber protein. As compared with Delta-24, Delta-24-RIVER induced EGFRvIII-selective cytotoxicity in U-87 MG isogenic cell lines and in tetracycline-inducible EGFRVIII expressing U-251 MG cells. Accordingly, by tittering the viral progeny and examining fiber protein expression in the above cells, we showed that the replication of this new construct also correlated with EGFRvIII expression. Consistently, immunohistochemistry staining of the adenoviral capsid protein hexon in the virus-treated tumors revealed that the virus replicated more efficiently in EGFRvIII-expressing U-87 MG.DeltaEGFR xenografts than in the tumors grown from U-87 MG cells. Importantly, treatment with Delta-24-RIVER prolonged the survival of animals with intracranial xenografts derived from U-87 MG.DeltaEGFR cells. Therefore, our results constitute the first proof of the direct targeting of a cancer-specific receptor using an oncolytic adenovirus.

Bacterial expression and kinetic characterization of the human monoamine-sulfating form of phenol sulfotransferase.

The cDNA for the human monoamine-sulfating form of phenol sulfotransferase (hM-PST) was isolated from a T47D human breast carcinoma lambda gt10 cDNA library, and the active enzyme was expressed in Escherichia coli. Expressed hM-PST was very similar to the brain, intestinal, and platelet forms of the enzyme in its physical, immunological, and kinetic properties. The ability of hM-PST to sulfate a number of xenobiotics was examined and compared with the bacterially expressed human phenol-sulfating form of PST (hP-PST). The translation product of the T47D hM-PST cDNA was 92% identical to that of liver hP-PST. Monoamine neurotransmittors, such as epinephrine and dopamine, were maximally conjugated at lower concentrations by expressed hM-PST (2 and 20 microM, respectively) than by hP-PST (1 and 1 mM, respectively). In contrast, simple phenols--such as p-nitrophenol, acetaminophen, and alpha-naphthol--were maximally conjugated at lower concentrations (4 microM, 20 microM, and 0.5 microM, respectively) by hP-PST than by hM-PST (1 mM, 1.5 mM, and 50 microM, respectively). Minoxidil was sulfated at similar rates and concentrations (7 mM) by both forms of PST. None of the estrogens or related compounds, such as beta-estradiol, 17 alpha-ethinylestradiol, diethylstilbestrol, equilenin, or genistein tested as substrates were sulfated by hM-PST; however, all of these compounds were substrates for hP-PST. As with hP-PST, the hydroxysteroids dehydroepiandrosterone and cortisol were not sulfated by hM-PST.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioluminescence imaging reveals a significant role for complement in liver transduction following intravenous delivery of adenovirus.

The effect of complement on transgene expression was evaluated in vivo and in vitro using mice lacking complement components. Complement component 3 (C3) deficient mice (C3-/-) and appropriate wild-type controls were intravenously injected with a replication incompetent, luciferase-expressing normal Ad5 (Ad5Luc1), or fibritin-fiber Ad5 (Ad5FFLuc1). Repeated, noninvasive bioluminescence imaging was conducted over 35 days. Our data show for the first time that C3 facilitates both short- and long-term hepatic expression of luciferase following systemic delivery. C3-/- mice showed significantly less (P < 0.05) luciferase expression in their liver than treatment-matched wild-type mice when 2.3 x 10(9) (Ad5Luc1) and 4.0 x 10(9) (Ad5Luc1 or Ad5FFLuc1) viral particles (v.p.) were infused. The maximal difference in luciferase activity between C3-/- and wild-type mice was 99-fold difference at 3 days for the 2.3 x 10(9) v.p. dose (Ad5Luc1), 35-fold at 13 days for the 4.0 x 10(9) v.p. dose (Ad5Luc1), and 22-fold at 13 days for the 4.0 x 10(9) v.p. dose (Ad5FFLuc1). Preincubation of Ad5Luc1 with wild-type, C1q-/-, or factor B (FB) deficient mouse sera for 5 min significantly (P < 0.05) increased transduction of mouse liver cells, as compared to preincubation with C3-/- sera or PBS. These results suggest the classical or alternate complement pathway enhances Ad5-mediated liver transduction.

Generation of recombinant adenovirus vectors with modified fibers for altering viral tropism.

To expand the utility of recombinant adenovirus vectors for gene therapy applications, methods to alter native viral tropism to achieve cell-specific transduction would be beneficial. To this end, we are pursuing genetic methods to alter the cell recognition domain of the adenovirus fiber. To incorporate these modified fibers into mature virions, we have developed a method based on homologous DNA recombination between two plasmids. A fiber-deleted, propagation-defective rescue plasmid has been designed for recombination with a shuttle plasmid encoding a variant fiber gene. Recombination between the two plasmids results in the derivation of recombinant viruses containing the variant fiber gene. To establish the utility of this method, we constructed a recombinant adenovirus containing a fiber gene with a silent mutation. In addition, we generated an adenovirus vector containing chimeric fibers composed of the tail and shaft domains of adenovirus serotype 5 and the knob domain of serotype 3. This modification was shown to alter the receptor recognition profile of the virus containing the fiber chimera. Thus, this two-plasmid system allows for the generation of adenovirus vectors containing variant fibers. This method provides a rapid and facile means of generating fiber-modified recombinant adenoviruses. In addition, it should be possible to use this system in the development of adenovirus vectors with modified tropism to allow cell-specific targeting.