NOVEL AD VECTORS AND APPLICATIONS

500. High-Level, Stable, In Vitro Gene initially we generated a porcine fetal retinal cell line (FPRT-HE1) Expression by a Foamy Virus/Adenovirus Hybrid that expresses E1 proteins of HAd5 and complements E1A function Vector of PAd3. FPRT-HE1 cell line was used for rescuing several PAd3 Rebecca A. Russell,1 Georges Vassaux,2 Myra O. McClure.1 vectors with deletions in E1A, E3 or both regions. All these vectors 1Jefferiss Research Trust Laboratories, Wright-Fleming Institute, were propagated to titers similar to wild type PAd3 in FPRT-HE1 2 cell line. A number of human or murine cancer cell lines were infected Imperial College, London, United Kingdom; Molecular Therapy Δ Laboratory, ICRF Molecular Oncology Unit, Imperial College, with PAd3 E1AE3-GFP (PAd3 vector with E1A and E3 regions London, United Kingdom. deleted and green fluorescent protein reporter gene in E1A region) or HAd5ΔE1E3-GFP (HAd5 vector with E1 and E3 regions deleted Retroviral vectors, capable of inducing long-term transgene and green fluorescent protein reporter gene in E1 region) and expression by virtue of their integration ability, are used as gene transduction efficiencies of the two vectors were monitored by flow delivery systems, but their development has been hindered by low cytometry. The results support our hypothesis that porcine transduction efficiencies, particularly in non-dividing cells. adenoviral vectors could be used as a promising supplement to Adenoviral vectors, in contrast, efficiently transduce both dividing human adenoviral vectors for gene therapy purposes. We are in the and non-dividing cells, but their genome remains episomal and is process of conducting an animal inoculation study in a murine model lost following repeated cell division. Hence, transgene expression is of breast cancer to explore the potential of eluding vector immunity transient. To capitalise on the advantageous characteristics of and toxicity by sequential administration of HAd and PAd vectors. retroviral and adenoviral vectors a number of MLV hybrid vectors have been generated. As alternative retroviral delivery systems, 502. The Expression of Two Genes Using a foamy viruses have much to offer, such as, an amphotropic host High-Capacity Adenoviral Vector range, a lack of associated disease in infected individuals, a larger Chi-Chi Liu,1 Shumei Zhong,1 Uma Singh,1 David L. Haviland,2 potential packaging capacity and an increased transduction efficiency Ba-Bie Teng.1 in non-dividing cells. These characteristics suggest that a prototype 1Research Center for Human Genetics, Inst. of Mol. Med., foamy virus (PFV)/adenovirus hybrid may out-perform the current University of Texas-Houston, Houston, TX; 2Research Center for adenovirus/MLV system in terms of in vivo potential. To this end, Immunology, Inst. of Mol. Med., University of Texas-Houston, an adenoviral/PFV hybrid vector has been produced which consists Houston, TX. of three adenoviruses encoding the PFV gag and pol genes (Ad- GagPoldelPacI), the PFV env gene (Ad-Env) and the PFV vector Atherosclerosis is a complex disease and multiple genes are genome encoding the enhanced green fluorescent protein (eGFP) involved in the progression of atherosclerotic lesion development. gene (Ad-MD9). Following adenoviral transduction, the target cells It is our goal to over-express multiple genes to achieve a therapeutic become transient PFV vector producing cells, releasing PFV vector effect on both prevention and treatment of this disorder. We used in situ for the stable infection of surrounding cells. Stable eGFP high-capacity adenovirus vector (pC4HSU provided by Merck & expression, observed for up to 38 days (seven passages) in cells Co. Inc.) to express simultaneously two cDNAs, the scavenger transduced with all three adenoviral vectors, was confirmed by PCR receptor class B Type I (SR-BI) and the apolipoprotein B mRNA to be the result of PFV, and not adenoviral, integration. In contrast, editing enzyme (Apobec1), driven by the CMV/intron A and the cells transduced with only the adenovirus encoding the PFV vector EF-1 alpha promoters, respectively. High titer stocks of this high- genome showed a marked decrease in eGFP expression at 16 days capacity adenoviral vector (HD-C2) were produced by using a Cre- post-transduction and did not contain integrated PFV vector. Thus, recombinase system. The number of viral particle was determined the PFV/adenovirus hybrid vector is capable of inducing long-term, by Real-time quantitative PCR. The average titer was 1 x high-level in vitro transgene expression. 1011particles/ml. SR-BI functions as a receptor for high density lipoprotein (HDL), 501. Porcine Adenoviral Vectors as Promising by mediating the selective uptake of the lipid moiety from HDL. Supplement to Human Adenoviral Vectors for Apobec1 is an essential enzyme that edits apoB mRNA to produce apoB48 protein. Overexpression of either SR-BI or Apobec1 has Gene Therapy been shown to regulate lipoprotein metabolism and influence the Dinesh Singh,1 Suresh K. Mittal.1 1 development of atherosclerosis. We have infected the COS cells Department of Veterinary Pathobiology, School of Veterinary with HD-C2 vector for 24, 48, and 72 h to demonstrate that both Medicine, Purdue University, West Lafayette, IN, United States. proteins (SR-BI and Apobec1) were biologically active when Human adenoviral (HAd) vectors offer numerous advantages as expressed simultaneously from the vector. The SR-BI and Apobec1 gene delivery vehicles for genetic therapy of a variety of diseases mRNAs were detected by Northern blot analysis at 24, 48, 72 h including cancer. Two major hurdles in putting these vectors to after infection. Western blot analysis of cell lysates demonstrated clinical use are: 1) preexisting and vector induced adenoviral immunity that both SR-BI and Apobec1 proteins were produced efficiently in and 2) vector toxicity. As a consequence of preexisting immunity in COS cells. The expression of the Apobec1 resulted in > 50% editing the majority of human population initial transduction with HAd of apoB mRNA as determined by an in vitro editing assay. By using vectors is often very difficult. In other cases development of strong fluorescently labeled Alexa-HDL and DiI-HDL, we demonstrated vector-specific immune response following first inoculation that the SR-BI expressed in the infected COS cells was able to bind drastically limits the vector uptake and thereby levels and the HDL and mediate efficient cellular uptake of HDL-associated lipids. duration of transgene expression after repeat administration. Next, we used the HD-C2 vector to infect HepG2 cells, human Moreover, administration of a higher vector dose to achieve aortic endothelial cells (HAOEC), and human aortic vascular smooth therapeutic levels of transgene expression almost always leads to muscle cells (AoSMC). Western Blot analysis of cell lysates shows acute inflammatory response and often fatal systematic toxicity. that SR-BI and Apobec1 were expressed efficiently in HepG2 cells, Use of nonhuman adenoviral vectors as an alternative to or in but the expression was relatively lower in HAOEC, and AoSMC conjunction with HAd vectors seems promising in overcoming these cells. Control HepG2 cells did not have editing activity and secreted limitations. In the present study we investigated the potential of full-length apoB100 only. HD-C2 infected HepG2 cells edited apoB replication defective porcine adenovirus type 3 (PAd3) vectors to transduce human and murine cancer cells in culture. As a prerequisite S196 Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy NOVEL AD VECTORS AND APPLICATIONS mRNA and secreted both apoB100 and apoB48 into the media. 504. New Adenoviral Packaging Cell Lines The secretion of apoB48 levels was time-dependent with a steady Expressing Pol, pTP, and the Recombinases Cre increase from 24 to 72 h. and Flp In conclusion, high-capacity adenoviral vectors can efficiently Michael J. Blankinship,1 Jeffrey S. Chamberlain.1 express two genes driven by two different promoters in various 1Neurology, University of Washington, Seattle, WA. cells types. Furthermore, the expressed proteins are biologically active. Thus, we speculate that by using high-capacity adenoviral Adenoviruses (Ad) lacking all viral genes (gutted adenoviruses), vectors we could deliver multiple genes to cells to provide therapeutic have several advantages over earlier generations of Ad vectors. effects to different target tissues at the same time. This novel gene Chiefly, these advantages lie in the increased cloning capacity and therapy approach could be applied to the simultaneous treatment decreased immune response resulting from deletion of the viral genes. and prevention of atherosclerosis. However, the growth of these viruses depends on the required viral gene products being supplied in trans. This complementation is 503. A Chimeric Helper-Dependent Gutless usually accomplished using a partially deleted helper virus in Ad5/35 Adenovirus Vector for Dual Adeno-/ conjunction with a packaging cell line, which together express the required gene products. However, this approach results in a final Retroviral Transduction of Hematopoietic Stem/ vector preparation containing both gutted and helper vectors. The Progenitor Cells helper level is usually decreased by flanking the packaging signal Helen Gharwan,1 Dmitry M. Shayakhmetov,1 Andre M. Lieber,1 with targets for a site-specific recombinase, i.e. Flp or Cre George Stamatoyannopoulos.1 recombinase. These modification results in the packaging signal 1 Medicine, University of Washington, Seattle, WA, United States. being excised when the virus is grown in a cell line expressing the Commonly used human adenovirus serotype 5 (Ad5) based respective recombinase. Here, we describe a series of new packaging vectors have proven to be inefficient for gene delivery into human cell lines for gutted adenoviruses based on the 911-cell line. hematopoietic stem cells, which are important targets for gene therapy Previously our lab and others have reported packaging cell lines of various hematopoietic diseases. Previously, we have shown the based on the 293-parent line expressing either Flp or Cre recombinase. superiority of chimeric adenoviral vectors containing the Ad35 fiber The 911 line produces a modest but significant increase in the amount within the Ad5 capsid (Ad5/35) for transduction of human CD34+ of adenovirus produced per cell when compared to a 293 derivative, cells, particularly the subsets with stem cell capacity. Since the in the range of 2-3 fold depending on the individual clone. These infection with first-generation adenoviral vectors is associated with lines have also been engineered to express the adenovirus polymerase cytotoxicity, we generated helper-dependent (HD) adenoviral (Pol) and pre-terminal protein (pTP) gene products. These genes vectors with Ad5/35 capsids. A HD Ad5/35 vector was produced allow for an increase in total virus titer, the ability to obtain gutted containing the ecotropic retroviral receptor gene sequence driven Ad preparations using minimal numbers of serial passages, as well from the PGK-promoter (HD Ad5/35-PecoR). Stable transgene as the option of using a helper virus deleted for Pol and/or pTP. For expression in target cells can be achieved by transient expression of selection against the helper virus, cell lines have also been engineered the retroviral receptor after a first transduction step with HD Ad5/ to express either Flp or Cre recombinase. These cell lines are capable 35-PecoR followed by a second transduction of the target cells with of excising approximately 99% of the packaging signals based on an ecotropic retroviral vector containing the gene of interest. The Southern and 24-hour burst assay analyses. These cell lines are titer of the adenoviral vector encompassed 5.7x1010 PFU/ml. Helper being compared to our previously reported C7 and C7-Cre cell lines virus contamination was less than 5% of viral genomes as determined as well as to a new 911 based cell line expressing both Cre and Flp by quantitative Southern Blot analysis. Preliminary experiments recombinase. These new reagents may significantly increase the were performed on a CD34+ growth factor-dependent human cell utility of gutted Ad vectors by facilitating preparation of high titer, line, Mo7e, using increasing doses of HD Ad5/35-PecoR (1, 5, 10, helper free gutted Ad vectors using multiply deleted helper viruses. 50, 100 and 400 PFU/ml). Twenty-four hours later the cells were transduced for 4 times every 12 hours with an ecotropic retrovirus 505. Comparative Analysis of the Genome expressing GFP at an MOI of 0.5. Forty-eight hours after the last Organization of Human Adenovirus 11, Species addition of retrovirus, the cells were analyzed for GFP-expression. B:2 and the Commonly Used Vector Ad5, Species We found that at low MOIs (1, 5 and 10 PFU/ml) of HD Ad5/35- C PecoR, increase of the adenoviral dose correlated with the increase Ya-Fang Mei,1 Johan Skog,1 Kristina Lindman,1 Göran Wadell.1 in the percentage of GFP-positive Mo7e cells (0.7% GFP-positive 1Department of Virology, Umeå University, Umeå, Sweden. cells at an MOI of 1; 2.4% at an MOI of 5 and 11.7% at an MOI of 10). At MOIs of HD Ad5/35-PecoR of 50 to 200 PFU/ml, the The genome of Ad11 possesses four early regions and six late percentage of GFP-expressing Mo7e cells reached a plateau of 35.5 regions, but Ad5 has been reported to have only five late regions. – 37.5%. Increasing the adenoviral MOI further to 400 PFU/ml, The genome of Ad11 was 1141 bp shorter than the Ad5 genome. In resulted in a reduced percentage of GFP-positive Mo7e cells (28.2%) the major late promoter region, the transcription elements are reflecting toxicity probably either derived from the presence of helper essentially homologous. However, the packaging region, VARNA, virus in the HD Ad5/35-PecoR preparation or from excessive ecoR- HVR region of hexon, and fiber region manifest pronouced differences. expression. A second analysis of GFP-expression 48 hours later More than 35 putative ORFs was identified in the Ad11 genome. In revealed an increase in the amount of GFP-positive cells (1.2% at an the comparison between Ad11 and Ad5 ORFs, the highest and MOI of 1; 3.8% at an MOI of 5; 18.4% at an MOI of 10; 45.5% at lowest homology was noted in the pTP and fiber gene, 85% and an MOI of 50, 52.5% at an MOI of 100; 69.7% at an MOI of 200 24.6 % , respectively. The E3 20..3, 20.6 K ORFs, L4 agnoprotein, and 49.3% at an MOI of 400) indicating stable integration of the and L6 agnoprotein were present in the Ad11 genome but could not retroviral DNA into the genome of the HD Ad5/35-PecoR-transduced found in Ad5 genome. A 11.6 K, cell death protein, in Ad5 E3 region cells. To evaluate our dual adeno-/retroviral system for ex vivo gene could not be identified in the Ad11 E3 region. Only the E3 10.3K therapy applications we have started with transduction studies on protein and L4 pVIII hexon associated protein shows identical size human mobilized peripheral blood CD34+ cells. The obtained data between Ad11 and Ad5. All other ORFs vary not only in will be presented and discussed. composition but also in size. Ad11 is suggested to have higher vector capacity than Ad5 vector, since Ad11 has a shorter fiber and Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts S197 Copyright © The American Society of Gene Therapy