Sheila Connelly

Development of adenovirus serotype 35 as a gene transfer vector.

While 51 human adenoviral serotypes have been identified to date, the vast majority of adenoviral vectors designed for gene transfer have been generated in the adenovirus serotype 5 (Ad5) backbone. Viral infections caused by Ad5 are endemic in most human populations and the majority of humans carry preexisting humoral and/or cellular immunity to Ad5 which may severely limit the use of Ad5-based vectors for gene therapy applications. To circumvent this preexisting Ad5 immunity, we have identified Ad35 as an alternative adenoviral serotype to which the majority of humans do not have neutralizing antibodies. Importantly, Ad35 can be grown to high titers with a low particle-to-PFU ratio. As a prerequisite for the development of Ad35 for use as a gene transfer vector, a genome organization map was constructed using the available Ad35 sequence information, and E1a-deficient Ad35 vectors encoding marker genes were generated. Ad35 biodistribution in mice was assessed following intravenous administration and compared with that of Ad5. Extremely low levels of Ad35 were detected in all organs evaluated, including liver, lung, spleen, and bone marrow, while Ad5 displayed high transduction of these organs. Due to the lack of Ad35 liver tropism, minimal hepatotoxicity was observed in mice treated with Ad35. Furthermore, the half-life of Ad35 in mouse blood was found to be two to three times longer than that of Ad5. These data suggest that either mice do not express the Ad35 cell surface receptor or that Ad35 does not efficiently transduce mouse cells in vivo following systemic delivery. Therefore, to begin to elucidate the Ad35 cell entry mechanisms, in vitro competition studies were performed. These data demonstrated that Ad35 cell entry is CAR independent, and may involve protein(s) expressed on most human cells.

Angiopoietin-2 enhances retinal vessel sensitivity to vascular endothelial growth factor.

Increased expression of vascular endothelial growth factor (VEGF) in the retina starting after postnatal day (P)7 results in neovascularization originating from deep retinal capillaries, but not those in the superficial capillary bed. Doxycycline was administered starting P0 to double transgenic mice with inducible expression of VEGF in the retina. These mice showed proliferation and dilation of superficial retinal capillaries, indicating that at this stage of development, the superficial capillaries are sensitive to the effects of VEGF. Angiopoietin‐2 (Ang2) is expressed along the surface of the retina for several days after birth, but by P7 and later, Ang2 is only expressed in the region of the deep capillary bed. In mice with ubiquitous doxycycline‐inducible expression of Ang2, in the absence of doxycycline, intravitreous injection of a gutless adenoviral vector expressing VEGF (AGV.VEGF) resulted in neovascularization of the cornea and iris, but no retinal neovascularization. After treatment with doxycycline to induce Ang2 expression, intravitreous injection of AGV.VEGF caused retinal neovascularization in addition to corneal and iris neovascularization. The retinal neovascularization originated from both the superficial and deep capillary beds. These data suggest that Ang2 promotes sensitivity to the angiogenic effects of VEGF in retinal vessels.

Characterization of the U3 and U6 snRNA genes from wheat: U3 snRNA genes in monocot plants are transcribed by RNA polymerase III.

We have demonstrated recently that the genes encoding the U3 small nuclear RNA (snRNA) in dicot plants are transcribed by RNA polymerase III (pol III), and not RNA polymerase II (pol II) as in all other organisms studied to date. The U3 gene was the first example of a gene transcribed by different polymerases in different organisms. Based on phylogenetic arguments we proposed that a polymerase specificity change of the U3 snRNA gene promoter occurred during plant evolution. To map such an event we are examining the U3 gene polymerase specificity in other plant species. We report here the characterization of a U3 gene from wheat, a monocot plant. This gene contains the conserved promoter elements, USE and TATA, in a pol III-specific spacing seen also in a wheat U6 snRNA gene characterized in this report. Both the U3 and the U6 genes possess typical pol III termination signals but lack the cis element, responsible for 3′-end formation, found in all plant pol II-specific snRNA genes. In addition, expression of the U3 gene in transfected maize protoplasts is less sensitive to α-amanitin than a pol II-transcribed U2 gene. Based on these data we conclude that the wheat U3 gene is transcribed by pol III. This observation suggests that the postulated RNA polymerase specificity switch of the U3 gene took place prior to the divergence of angiosperm plants into monocots and dicots.

Intraocular gutless adenoviral-vectored VEGF stimulates anterior segment but not retinal neovascularization

Vascular endothelial growth factor (VEGF) and insulin‐like growth factor‐1 (IGF‐1) have been implicated as important stimulatory factors for retinal neovascularization. In this study, we used intraocular gene transfer with gutless adenoviral (AGV) vectors to determine the effect of increased intraocular expression of VEGF, IGF‐1, or sphingosine kinase (SPK), which produces sphingosine‐1‐phosphate, another angiogenic factor. Retinal neovascularization did not occur from intravitreous AGV‐vectored VEGF, IGF‐1, SPK, or combined VEGF and IGF‐1, except occasionally adjacent to the retinal penetration site from the injection. However, corneal and iris neovascularization occurred after 2 weeks in all eyes injected with AGV.VEGF, but not those injected with only AGV.IGF‐1 or AGV.SPK. These data suggest that the superficial capillary bed of the retina is relatively insensitive to VEGF, IGF‐1, or SPK in adult mice, except when combined with retinal trauma. However, AGV‐vectored VEGF is sufficient to consistently cause severe corneal and iris neovascularization. This provides a model for anterior segment neovascularization, which unlike previous models is relatively inexpensive and is not plagued by spontaneous regression, and therefore, may be useful for identification of new treatments.

Differential expression of U5snRNA gene variants in maize (Zea mays) protoplasts

The small nuclear ribonucleoprotein particles U1, U2, U4/U6 and U5 participate in the removal of introns from pre-messenger RNAs in the nucleus. Three genes encoding U5snRNAs, the RNA moiety of U5snRNPs, have been isolated from maize. As in other plant UsnRNA gene families the three maize U5snRNA genes exhibit sequence variation. Two of the gene variants (MzU5.1 and MzU5.2) are clearly expressed after transfection into maize leaf protoplasts while the third gene variant (MzU5.3) is expressed at very low levels. These different levels of expression cannot be directly correlated with sequence changes in the highly conserved Upstream Sequence Element (USE) required for expression of Arabidopsis UsnRNA genes nor with differential stability of the U5snRNA transcripts. Further sequence elements may therefore have a role in regulating maize UsnRNA gene expression.

Characterisation and expression of a maize U3 snRNA gene.

We have used a probe encoding a U3snRNA gene of Arabidopsis to isolate maize U3snRNA genomic sequences. Of two clones sequenced, one encodes a single U3 gene which has been shown to be expressed in transfected maize protoplasts. The second clone encodes a U3 related sequence which appears to be an RNA-mediated pseudogene.