Senior Scientist
,
Regenerative Medicine
, Ottawa Hospital Research Institute
PREVIOUS STUDIES
The Cre/loxP system for generating hdAd's. In 1996, we developed the Cre/loxP system for the generation of hdAd, which involves the use of a helper virus with a packaging signal flanked by loxP sites (Parks et al. 1996,Proc. Natl. Acad. Sci. U.S.A. 93:13565-13570). Upon infection of a 293-derived cell line that expresses the Cre recombinase, the packaging signal is excised rendering the helper virus DNA unpackageable. The helper virus is still able to replicate and provide in trans all of the functions required for replication and packaging of the hdAd, and results in vector stocks with dramatically reduced quantities of helper virus (typically less than 0.1%).
Adenovirus DNA size constraints . Using the Cre/loxP system and a series of hdAd between 15.1 and 33.6 kb, we determined that Ads had a lower limit to efficient DNA packaging of about 75% of the wildtype genome length (Parks and Graham 1997, J. Virol. 71:3293-3298). Smaller viral genomes tended to rearrange resulting in a final size greater than 30 kb (e.g. the 15.1 kb vector was recovered as a covalent dimer of 30.2 kb). Thus, in order to generate stable hdAd's, we must construct the vectors of a final size greater than about 28 kb, but less than 37 kb (the upper limit for Ad DNA packaging). This requirement for a minimum hdAd vector size implies that in many cases additional "stuffer" DNA must be included in the hdAd. We determined that the nature of the stuffer DNA can have important effects on the functioning of the vector (Parks et al. 1999, J. Virol. 73:8027-8034). For example, a hdAd which contained 22 kb of bacteriophage lambda DNA showed poor expression in vitro and in vivo, and elicited the formation of cytotoxic T-lymphocytes (CTL), to peptides produced from the lambda DNA, in immunized mice. In contrast, a similar hdAd with human HPRT DNA as stuffer showed significantly improved expression and did not elicit HPRT-specific CTL.
Adenovirus serotype switching. Ad administration to immunocompetent individuals results in the formation of anti-Ad neutralizing antibodies which are a significant barrier to vector readministration. Using sequential treatment of mice with hdAd vectors of alternative serotype, we could effectively readminister hdAd vectors (Parks et al. 1999, Gene Therapy 6:1565-1573). A key feature of the hdAd system is that the serotype of the vector is dependent on the helper virus and, thus, a series of genetically identical hdAds of different serotypes could be generated simply by changing the helper. Thus, should transgene expression levels decrease over time, the use of hdAds of alternative serotypes will permit readministration of a vector with the identical genotype.
HdAd vector efficacy. In collaboration with researchers at Baylor College of Medicine (Houston, Texas), we have shown that hdAd produced with the Cre/loxP system provide high levels of expression of the human alpha-1-antitrypsin gene for considerably longer (>1 year) than a first generation Ad vector (fgAd) in mice (Scheidner et al. 1998, Nature Genetics. 8:180-183), and in non-human primates (Morral et al. 1999, Proc. Natl. Acad. Sci. USA 96:12816-12821). Furthermore, histopathological examination revealed that mice treated with the hdAd had essentially normal liver morphology even at very high vector doses (Morral et al. 1998, Human Gene Therapy. 9:2709-2716), whereas animals treated with a first generation vector showed significant acute and chronic liver injury. Similar results were observed by investigators at Merck Research Laboratories ( West Point , Pennsylvania ) after gene transfer in a leptin knockout mouse model (Morsy et al. 1998, Proc. Natl. Acad. Sci. USA 95:7866-7871). Taken together, these results indicate that hdAd deleted of all coding sequences are an efficient gene delivery vehicle.
HdAd for cancer therapy. The human Ad5 early region 1A (E1A) proteins have potent antitumor effects, due to their ability to reprogram oncogenic signaling pathways in tumor cells. The success of E1A antitumor therapy in animal models has led to its use in phase I and phase II clinical trials, where liposome-based delivery vehicles are being used to deliver a plasmid encoding E1A. To increase the efficiency of E1A delivery to tumors, we developed a hdAd deleted of all viral protein coding sequences with the exception of E1A,designated hdAd-E1A (Hubberstey et al. 2002, Cancer Gene Therapy 9:321-329). In culture, this vector mediated high-level expression of E1A gene products. A549 cells, a human lung adenocarcinoma cell line, infected with hdAd-E1A showed a reduced proliferative capacity in adherent culture, and the ability to form colonies in soft agarose was completely abolished. A549 cells treated with hdAd-E1A showed approximately 10-fold greater sensitivity to the chemotherapeutic drug cisplatin. These data indicate that the use of hdAd provides a simple and effective method to deliver E1A to cancer cells.
Immune responses to Ad vectors. The earliest events in the cells response to Ad infection are initiated by binding of the viral capsid to the cell, and culminate with the activation of the innate immune response. After vector delivery, the Ad capsid proteins immediately activate the expression of cytokines and chemokines within the infected cell. Since the capsid of hdAd and fgAd are identical, these early, capsid-driven responses should be identical for the two classes of vector. In collaboration with Dr. Daniel Muruve ( University of Calgary ), we have shown this to be true (Muruve et al. 2004, J Virol 78:5966). Delivery to mice of hdAdSEAP (SEAP - a murine secreted alkaline phosphatase that we cloned and characterized, Maelandsmo et al. 2004, J Gene Medicine, in press) induced an early (~6 hr) expression of inflammatory cytokine/chemokine genes in the liver, including IP-10, MIP-2 and TNF- a , identical to that induced by fgAdSEAP. All three vectors induced the recruitment of CD11b positive leukocytes (monocytes, NK cells, neutrophils) within hours of administration. fgAdSEAP also induced a second phase of liver inflammation, consisting of inflammatory gene expression and CD3 positive lymphocytic infiltrates 7 days post-transduction. In contrast, beyond 24 hours no infiltrates or expression of inflammatory genes were detected in the livers of mice receiving hdAdSEAP. These results demonstrate that hdAd vectors induce an intact innate response in vivo, but that adaptive immune responses are attenuated, likely contributing to the enhanced efficacy of hdAd-mediated gene therapy.
Ad vectors for muscle-directed gene therapy. Ads do not transduce mature muscle very efficiently due to low levels of the natural viral primary receptor, the Coxsackie and adenovirus receptor (CAR), on the surface of adult muscle cells. We addressed whether incorporation of a poly-lysine (pK) or arginine-glycine-aspartic acid (RGD) placed in the H-I loop of fiber can improve hdAd transduction of mature muscle cells (Bramson et al. 2004, Human Gene Therapy 15:179). pK binds to heparin sulphate proteoglycans prevalent on many different cell types (including muscle) and the RGD motif will redirect Ad binding to specific integrins present on muscle cells. Incorporation of the pK motif into fibre resulted in up to a 21-fold enhancement of muscle cells in culture, whereas incorporation of the RGD motif resulted in only a 70% increase. Although hdAdpK also enhanced infection of mature mouse muscle in vivo, hdAdRGD did not. Our results demonstrated that hdAd with modified cell tropism can lead to improved transduction of adult muscle cells, but that other barriers likely also contribute to poor transduction of muscle by Ad, aside from low levels of the viral receptor on muscle cells.
In the study described above, in vitro infection with the fibre-modified vectors was much more efficient than in vivo. We hypothesized that physical barriers, such as the basal lamina, may represent significant obstacles to Ad, and prevent the virus from physically interacting with the muscle cell membrane. Using our pIX-GFP tagged virus (described below), we showed that, shortly after injection of mouse muscle, Ad vector seems to accumulate in pockets between the muscle fibers that are created as a result of the injection process (Meulenbroek et al. 2004, Molecular Therapy 9:617). Moreover, although Ad does disperse from the site of injection along the length of adjacent fibers, in general, it does not travel far from the injection site. Failure to efficiently disperse in the muscle may be one reason why Ad does not infect muscle well, even when the virus is targeted to receptors that are prevalent on muscle cells.
Ad capsid protein IX (pIX). As part of our ongoing efforts to develop new methods for the preparation of hdAd vectors, we became interested in the Ad capsid pIX. pIX associates with the hexons that make up the facets of the icosahedron and, deletion of pIX results in virions that are heat labile, with capsids that can accommodate only 35 kb of viral DNA (~97% of the wildtype genome). Thus, deletion of pIX provides a means of selecting for virions that contain viral DNA that is less than the size of the wildtype genome. This observation was the basis of a new method we developed to selectively package hdAd DNA. Since this represents a physical barrier to helper virus DNA packaging rather than an enzymatic restriction, such as a Cre mediated one (Parks et al. 1996,Proc. Natl. Acad. Sci. U.S.A. 93:13565-13570), we anticipated that such a system might lead to dramatically reduced levels of helper virus contamination if not complete elimination of the helper virus from hdAd stocks.
A pIX- helper virus (~37.3 kb) was easily grown on complementing 293pIX cells. Upon infection of non-complementing cells, this virus was not capable of forming infectious virions, but provided replicative and packaging functions for propagation of a 30 kb hdAd (Sargent et al. 2004, Gene Therapy 11:504). The pIX- helper virus was effective at amplifying a hdAd and, in combination with Cre-mediated excision in the viral packaging signal, resulted in a 1000-fold reduction in helper virus contamination in hdAd stocks compared to Cre/lox alone, as determined by plaque assay. However, a large amount of the 37.3 kb helper DNA was also packaged into the pIX-deleted virions, but these virions were incapable of establishing productive infections in plaque assays, for reasons which are still unclear. These data suggest that, although further studies are necessary to characterize the nature of the defective helper virions formed in this system, deletion of pIX from the helper virus genome does provide an effective method to prevent recovery of functional helper virus during hdAd amplification.
In addition to its role as a structural protein, pIX has been implicated as a transcriptional activator, and in transient transfection assays was shown to enhance expression from the Ad early region 1A (E1A), E4 and major late promoters (MLP). If this activity is crucial for Ad replication, it suggested that the reason our helper virus did not form plaques in the Cre/loxP/pIX helper-dependent system described above was the lack of pIX. Therefore, we undertook a study to examine the role of pIX's ability to activate transcription during Ad replication (Sargent et al. 2004, J Virol 78:5032). We used hdAd vectors, which were deleted of all Ad protein coding sequences with the exception of E1A, and with capsids that either contained or lacked pIX, to show that pIX derived from decapsidation of the infecting virion does not influence expression of E1A. Similarly, expression of pIX from the Ad virus genome did not alter the expression levels of E1A. Viruses that were deleted of pIX showed a small 3-fold reduction in virus yield and expression of late genes compared to a similar virus which encoded pIX. Thus, although pIX can affect transcription from a variety of viral promoters, it does not appear to play a significant role in activation of Ad promoters during normal Ad replication.
The goal of Ad targeting is to ablate the natural Ad tropism, thereby reducing Ad infection of non-target tissues, while concomitantly redirecting virus attachment to a unique receptor found on the cell type of interest. Since pIX is exposed on the surface of the virion, it may represent a new capsid protein to which large targeting ligands can be attached. As proof of principle, we generated a pIX-green fluorescent protein (GFP) fusion protein, and characterized these novel vectors (Meulenbroek et al. 2004, Molecular Therapy 9:617). The fusion protein was efficiently incorporated into the Ad capsid and, in tissue culture, translocation of Ad/pIX-GFP from the outside of the cell to the nucleus could be visualized using fluorescence microscopy. We also could track the virus after injection in the tibialis anterior muscle of mice. Our ability to attach GFP to the Ad virion, through fusion to pIX, provides a valuable tool for virus tracking in vitro and in vivo. Moreover, our data indicates that pIX can be used as a platform to anchor proteins to the Ad capsid, such as large ligands for cell-type specific targeting of the vector.
General Campus
(see 
