Significant alterations of biodistribution and immune responses in Balb/c mice administered with adenovirus targeted to CD40(+) cells.

CD40 ligation has been shown to promote antigen-presenting functions of dendritic cells, which express CD40 receptor. Here we reported significantly altered biodistribution and immune responses with the use of CD40-targeted adenovirus. Compared with unmodified adenovirus 5, the CD40-targeted adenovirus following intravenous administration (i.v.) resulted in increased transgene expressions in the lung and thymus, which normally do not take up significant amounts of adenovirus. Intradermal injection saw modified adenovirus being mainly processed in local draining lymph nodes and skin. Following intranasal administration (i.n.), neither unmodified nor targeted viruses were found to be in the liver or spleen, which predominantly took up the virus following i.v. administration. However, inadvertent infection of the brain was found with unmodified adenoviruses, with the second highest gene expression among 14 tissues examined. Importantly, such undesirable effects were largely ablated with the use of targeted vector. Moreover, the targeted adenovirus elicited more sustained antigen-specific cellular immune responses (up to 17-fold) at later time points (30 days post boosting), but also significantly hampered humoral responses irrespective of administration routes. Additional data suggest the skewed immune responses induced by the targeted adenoviruses were not due to the identity of the transgene but more likely a combination of overall transgene load and CD40 stimulation.

An adenovirus vector with a chimeric fiber incorporating stabilized single chain antibody achieves targeted gene delivery.

Adenovirus (Ad) vectors are of utility for many therapeutic applications. Strategies have been developed to alter adenoviral tropism to achieve a cell-specific gene delivery capacity employing fiber modifications allowing genetic incorporation of targeting motifs. In this regard, single chain antibodies (scFv) represent potentially useful agents to achieve targeted gene transfer. However, the distinct biosynthetic pathways that scFv and Ad capsid proteins are normally routed through have thus far been problematic with respect to scFv incorporation into the Ad capsid. Utilization of stable scFv, which also maintain correct folding and thus functionality under intracellular reducing conditions, could overcome this restriction. We genetically incorporated a stable scFv into a de-knobbed, fibritin-foldon trimerized Ad fiber and demonstrated selective targeting to the cognate epitope expressed on the membrane surface of cells. We have shown that the scFv employed in this study retains functionality and that stabilizing the targeting molecule, per se, is critical to allow retention of antigen recognition in the adenovirus capsid-incorporated context.

Mesothelin-mediated targeting of adenoviral vectors for ovarian cancer gene therapy.

Adenoviruses (Ads) are efficient gene transfer vehicles, but Ad-mediated gene therapy for ovarian cancer remains limited in vivo by inefficient and nonspecific gene transfer. Mesothelin (MSLN), a cell surface glycoprotein, is overexpressed in ovarian cancer but not in normal tissues except mesothelial cells. Therefore, MSLN is an attractive candidate for transcriptional and transductional targeting in the context of ovarian cancer gene therapy. We evaluated the expression of MSLN mRNA and MSLN surface protein in ovarian cancer cells. Ads containing the MSLN promoter driving reporter gene expression were created and tested in ovarian cancer cell lines and purified ovarian cancer cells isolated from patients. To evaluate transductional targeting, we used an Ad vector containing an Fc-binding domain within the fiber protein, which served as a docking domain for binding with anti-MSLN immunoglobulins. Both RT-PCR and flow cytometry revealed high MSLN gene and protein expression in ovarian cancer cells. The MSLN promoter was activated in ovarian cancer cells, but showed significantly reduced activity in normal control cells. Transductional targeting of Ads via anti-MSLN antibody increased transgene expression in ovarian cancer cells. This report describes the use of MSLN for transcriptional as well as transductional targeting strategies for ovarian cancer gene therapy.

Genetic targeting of an adenovirus vector via replacement of the fiber protein with the phage T4 fibritin.

The utility of adenovirus (Ad) vectors for gene therapy is restricted by their inability to selectively transduce disease-affected tissues. This limitation may be overcome by the derivation of vectors capable of interacting with receptors specifically expressed in the target tissue. Previous attempts to alter Ad tropism by genetic modification of the Ad fiber have had limited success due to structural conflicts between the fiber and the targeting ligand. Here we present a strategy to derive an Ad vector with enhanced targeting potential by a radical replacement of the fiber protein in the Ad capsid with a chimeric molecule containing a heterologous trimerization motif and a receptor-binding ligand. Our approach, which capitalized upon the overall structural similarity between the human Ad type 5 (Ad5) fiber and bacteriophage T4 fibritin proteins, has resulted in the generation of a genetically modified Ad5 incorporating chimeric fiber-fibritin proteins targeted to artificial receptor molecules. Gene transfer studies employing this novel viral vector have demonstrated its capacity to efficiently deliver a transgene payload to the target cells in a receptor-specific manner.

[Characterization of rat lines with normotensive and hypertensive status using genomic fingerprinting]. / Kharakteristika linii krys s normotenzivnym i gipertenzivnym statusom metodom genomnogo fingerprintinga.

The properties of normotensive and hypertensive rat lines were investigated by the DNA fingerprinting method using a multilocus micro-satellite (CAC)5 probe. The HaeIII and HinfI restriction endonucleases were found to be the most informative enzymes in this case. The high genetic homogeneity of the ISIAH line, a rat line with inherited stress-sensitive arterial hypertension created at the Institute of Cytology and Genetics, was demonstrated. The lack of intralinear polymorphism was also typical to Japanese SHR rats with spontaneous arterial hypertension. Normotensive WAG rats had identical fingerprinting patterns, while the relative intensity of some bands was different. The outbred normotensive Wistar line maintained at the Institute, appeared to carry 30% polymorphic alleles. Hypertensive lines differed from the normotensive by a number of genetic markers.

[Use of a synthetic oligonucleotide (CAC)5 in the genomic "dactyloscopy" method]. / Izpol'zovanie sinteticheskogo oligonukleotida (TsATs)5 v metode genomnoi "daktiloskopii".

Potentialities of a chemically synthetized oligonucleotide of a certain structure in the genomic "dactyloscopy" method were under study. A random sample analysis has demonstrated a high resolving power of this variant of the method. Arguments in favor of introducing this approach in practical forensic medical direct identification of biologic material are presented.

[Nature of the endogenous retrovirus-like particles of the rat liver]. / Issledovanie prirody éndogennykh retrovirusopodobnykh chastits pecheni krys.

Previously virus-like particles (VLP) with properties resembling retroviruses were isolated from the liver of Wistar rats. Molecular hybridization and CRIA test were used for further analysis of the VLP. The CRIA method showed that VLP preparation lacked antigenic determinants of the major internal protein of C-type virus. By the dot hybridization technique no homology was detected between VLP and Mo-MuLV DNA, however VLP RNA was found to be homologous to IAP (interstitial A particles) DNA of mice. VLP proviral DNA was detected in the rat genome by the blot hybridization technique. Thus, it was concluded that VLP resemble IAP. A possible role of rat IAP expression is discussed.

Reverse transcriptase of rat liver associated with the endogenous retrovirus related to the mouse intracisternal A-particles.

Reverse transcriptase activity was found in rat liver enclosed in virus-like particles. Through hybridization with DNA probes of A- and C-type retroviruses and with the help of electron microscopy the virus-like particles have been identified as endogenous retroviruses related to the mouse intracisternal A-particles. Blot hybridization revealed the provirus DNA in the rat genome. The enzyme was isolated from the virus-like particles, purified and characterized. The main properties of the enzyme resemble those of the mammalian retrovirus reverse transcriptase.

[Changes in the RNA-dependent DNA-polymerase activity of retrovirus-like particles in the rat liver during postnatal ontogeny]. / Izmenenie RNK-zavisimoi DNK-polimeraznoi aktivnosti retrovirusopodobnykh chastits pecheni krys v protsesse postnatal'nogo ontogeneza.

The RNA-dependent DNA-polymerase activity was studied in the postmicrosomal fraction and in the microsomal sediment of the liver of the newborn and adult Wistar rats. In the microsomal sediment of 4-6 day old rats the RNA-dependent DNA-polymerase activity was approximately by one order of magnitude higher than in that of 2 week old and adult rats. In the postmicrosomal fraction of 3 day old rats the RNA-dependent DNA-polymerase activity was also higher, but only by 30-35%, than in that of animals of the other age groups. Particles with density of 1.17 g/ml were found in the microsomal sediment of all studied animals. The characteristic morphology, sensitivity of the particle RNA-dependent DNA-polymerase activity to RNAse and the presence of reverse transcriptase allow for these particles to be referred to as retroviruses. A suggestion is put forward that the high intensity of reverse transcription during the early postnatal period can be due to still continuing processes of cell differentiation and enzymatic imprinting.

[Relation of RNA-dependent DNA-polymerase from the rat liver with virus-like particles]. / Sviaz' RNK-zavisimoi DNK-polimerazy iz pecheni krys s virusopodobnymi chastitsami.

RNA-dependent DNA-polymerase activity was found in the 165 000 g supernatant and pellet of the postmitochondrial rat liver fraction. Further fractionation of the 165 000 g pellet in the linear sucrose gradient (20-50%) showed that RNA-dependent DNA-polymerase activity was distributed between fractions with densities 1.18-1.19 g/ml and 1.09-1.1 g/ml. In the fractions with 1.18-1.19 g/ml density the enzymic activity could be detected only after Triton X-100 treatment and disappeared after the incubation with pancreatic ribonuclease A. Triton X-100 treatment of the 165 000 g supernatant and the fractions with density 1.09-1.1 g/ml did not increase further the enzymic activity. Electron microscopy revealed in the 1.18 g/ml fraction virus-like particles resembling retroviruses of A and C type. In the light peak "non-mature" virus-like particles were found. The 165 000 g supernatant devoid of virus-like particles contained free RNA-dependent DNA-polymerase activity. The virus-like particles of both types seem to be endogenous rat retroviruses serving as a source of the particular and free reverse transcriptase in the rat liver.