411 A Helper Dependent Adenovirus Epstein Barr Virus Hybrid System that Stably Transforms Cells in Vitro with High Efficiency Molecular Therapy �������� ��� ���� ���������������� �������� ���� ������©[.]
VECTOR GENOME BIOLOGY positive for vector sequences in the period immediately following vector injection Adult animals (n=27) were injected at doses ranging from x 10E11 to x 10E13 vg/kg and semen samples were collected weekly DNA obtained from total semen, motile, and non-motile sperm fractions were assayed by the same sensitive PCR developed for the clinical study We found that rabbits uniformly showed a positive signal in serum at days post injection Moreover, there was a dose-dependent increase in the likelihood of finding vector sequences in semen DNA Thus, animals injected at x 10E11 vg/ kg were initially positive but cleared by week following injection However, animals injected with a dose of x 10E12 vg/kg or x 10E13 vg/kg required 8- 13 weeks to clear Semen fractions enriched for motile sperm were transiently positive and the clearance was faster than for total semen specimens These data suggest that clearance of vector sequences from semen DNA eventually occurs and that the kinetics of clearing is dose-dependent Findings from the clinical study demonstrated PCR positivity in the semen samples from all subjects enrolled (AAV vector dose ranging from x 10E10 to x 10E12 vg/kg) when samples collected at day following injection were assayed To address the duration of shedding of infectious AAV particles into the semen, we carried out an AAV infectivity assay using serial semen samples collected from injected rabbits The results demonstrated that even at the earliest time point assayed (day 7) no infectious AAV particles were detected in the semen as was also the case for semen samples collected at later time points Animals followed for over a year (more than spermatogenesis cycles) not reveal any subsequent recurrence of vector sequences in the semen, which suggests that no early spermatogonial precursors were affected by vector In addition, for subjects below 40 years of age, the period of vector shedding was 46 weeks, considerably shorther than that observed for of older subjects (8-12 weeks) Together these results suggest that the risk of persistent PCR positivity of semen DNA for vector sequences is low, and that AAV infectious particles are not present even in samples collected at early time points The data also suggest that the duration of vector shedding may be shorter for younger patients Dr Couto, Dr Kay, Ms Chew, Dr Zhen and Dr Sommer have equity at Avigen, Inc injection, is less well defined and likely involves de novo cellular transcription and amplification of cytokine release and the systemic inflammatory response Several days after Ad administration chronic toxicity may arise It is primarily mediated by the host adaptive immune response to de novo production of antigens In early generation Ad vectors, the primary antigens derive from viral gene expression, though potential neo-antigenicity of the therapeutic transgene may also contribute Indicators of vector-associated toxicity include elevation of cytokine levels, including IL-6, and increase in plasma levels of liver transaminases and reduction in circulating platelet numbers We evaluated these parameters to compare early and intermediate toxicity associated with systemic administration of equal doses of first generation (FG-Ad) and helper-dependent (HD-Ad) vectors containing the same expression cassette in C57BL/ 6J mice We confirmed that FG-Ad and HD-Ad have a different effect on chronic toxicity but induce a similar acute toxicity profile in the first 12 hours after systemic administration In order to further elucidate the genetic determinants of this host response we administered Ad vectors into mice compromised in different immune signaling pathways Our data suggest an involvement of an early involvement of a TNF-α-mediated response to adenoviral capsid proteins as a possible event leading to the dose-dependent thrombocytopenia associated with adenoviral vector administration Furthermore we exclude a significant contribution of interferon-γ and perforin-mediated pathways in acute and intermediate toxicity Finally we determine that mast cell depletion does not improve platelet counts measured 72 hours after Ad vectors injection Importantly we show that pretreatment with anti TNF-α antibody reduced early toxicity associated with vector administration as shown by decreases in IL-6 levels and thrombocytopenia, without concurrent effect on efficiency of transduction or IL-12 levels Therefore, the use of HD-Ad vectors in combination with pharmacological modulation of TNF-α may improve the therapeutic index of human gene therapy trials NOVEL AD VECTORS - AD RECEPTORS Oliver Dorigo,1 Jose S Gil,1 Sean Gallaher,1 Brent Tan,1 Arnold J Berk.1 Molecular Biology, UCLA, Los Angeles, CA, United States 410 Determinants of and Pharmacologic Modulation of Acute Toxicity Associated with Systemic Administration of First Generation and Helper-Dependent Adenoviral Vectors Gabriele Toietta,1 Viraj P Mane,1 Lucio Pastore,2 Milton J Finegold,3 Philip Ng,1 Arthur L Beaudet,1 Brendan Lee.1,4 Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States; 2Dipartimento di Biochimica e Biotecnologie Mediche and CEINGE-Biotecnologie Avanzate, Universita’ degli Studi di Napoli Federico II, Naples, Italy; Pathology, Baylor College of Medicine, Houston, TX, United States; 4Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX, United States Understanding how systemic administration of adenoviral (Ad) vectors elicits an acute host response is critical for human clinical gene therapy Toxicity associated with systemic Ad administration is the result of a multi-phasic process Early, acute toxicity is related to viral interaction with target cells and/or consequent vectors entry with viral uncoating This process occurs rapidly within minutes to hours of vector administration The activation of cytokines and thrombocytopenia associated with acute toxicity may result, at high doses, in disseminated intravascular coagulation and multi-organ damage Intermediate toxicity, occurring from hours to days after S162 411 A Helper Dependent Adenovirus-Epstein Barr Virus Hybrid System that Stably Transforms Cells in Vitro with High Efficiency Epstein Barr Virus (EBV) episomes are stably maintained in proliferating cells due to Epstein Barr Nuclear Antigen-1 (EBNA-1) protein mediated segregation and replication once per cell division We have previously reported the delivery of an EBV episome using an adenovirus-EBV hybrid system (Tan et al, J Virol, 73(9), p.7582) This system utilizes Cre/loxP mediated recombination of the linear adenoviral genome into a circular episome that stably transforms canine D17 cells However, maximum transformation efficiency required doses of the E1-deleted adenoviral vectors that caused significant toxicity to infected cells In the present study, we have utilized a binary helper dependent adenoviral (HDA) vector system to deliver an EBV episome using Cre/loxP recombination Our approach included the design of a HDA for the expression of Cre (HDA.Cre) and a second HDA for the delivery of the EBV episome (HDA.PAC and HDA.CFP) All vectors contain an expression cassette for yellow fluorescent protein for titration The sequences for the EBV episome - including the EBV family of repeats, an EBNA-1 expression cassette, the transgenes for puromycin acetyltransferase (PAC) or cyan fluorescent protein (CFP), as well as a 19kb human DNA sequence for replication - were flanked by loxP sites Upon co-infection of canine D17 and human HeLa cells with HDA vectors in vitro, the loxP vectors underwent Cre induced recombination and generation of an EBV episome as assayed by Southern Blot analysis Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy NOVEL AD VECTORS - AD RECEPTORS Using a colony-forming assay in puromycin selection medium, we demonstrated stable transformation of up to 61% of D17 cells after co-infection with HDA.Cre and HDA.PAC Furthermore, cell viability at the MOI necessary to achieve this transformation efficiency was significantly better compared to the E1-deleted system (66.2% vs 5.4%) Due to reduced cytotoxicity, the HDA-EBV system was found to be significantly more efficient compared to the E1-deleted system Stable transformation was also achieved in 16% of co-infected HeLa cells without significant cytotoxicity To further optimize the binary HDA-EBV system, we used CFP as marker for recombination Due to different excitation and emission spectra, CFP as expressed on the episome can be distinguished for YFP on the linear vector using fluorescence microscopy Interestingly, variations of the MOI for HDA.Cre and HDA.CFP resulted in significantly different recombination efficiencies calculated as percentage of CFP positive cells We found that co-infection of HDA.Cre at MOI=10 (titered by YFP transduced units) and HDA.CFP at MOI=30 resulted in 40-50% CFP positive cells after days A further increase of the MOI for HDA.Cre surprisingly resulted in a reduction of recombination efficiency, possibly due to competition between the two vectors for cell entry In contrast, an increase of the MOI for HDA.CFP resulted in a slightly greater percentage of recombination but also a decrease in cell viability In summary, we have constructed a HDA-EBV hybrid system capable of stably transforming mammalian cells in vitro Due to significantly reduced cytotoxicity, this system was found to be superior to the previously reported E1 deleted adenovirus mediated EBV delivery system 412 Z Domain-Modified First-Generation and High-Capacity Adenoviral Vectors for AntibodyMediated Targeted Gene Transfer Christoph Volpers,1 Volker Biermann,1 Christian Thirion,2 Stefanie Hussmann,3 Helmut Kewes,1 Andreas Herrmann,3 Hanns Lochmueller,2 Stefan Kochanek.1 Center for Molecular Medicine (ZMMK), University of Cologne, Cologne, Germany; 2Gene Center, University of Munich, Munich, Germany; 3Cardion AG, Erkrath, Germany In order to target adenoviral (Ad) vectors to cell types lacking or expressing low levels of the Ad receptor, target-specific peptide ligands have been genetically incorporated into the capsid, fiber proteins from Ad serotypes with different tropism have been inserted, and bispecific recombinant or chemically engineered adaptor molecules have been used However, small, high-affinity binding peptide ligands suitable for capsid incorporation are available only for few cell surface molecules, and all these strategies have to be specifically tailored for each respective target receptor or cell type Therefore, we have developed an efficient and highly versatile new strategy allowing the direct use of monoclonal antibodies against cell surface antigens for targeting of Ad vectors A full-length Zdomain (Z59; 59 amino acids) or a shorter version of it (Z33; 33 amino acids), IgG-binding domains derived from staphylococcal protein A, were inserted into the Ad fiber protein Both modified fiber proteins retained the ability to assemble into trimers, they bound IgG with high affinity as demonstrated by ELISA and surface plasmon resonance measurements, and they were efficiently incorporated into vector particles Pull-down experiments with IgGSepharose and competition ELISA confirmed IgG-binding activity of the Z-modified vectors By preincubation with different concentrations of a monoclonal antibody directed against epidermal growth factor receptor (anti-EGFR), transduction efficiencies of the Z33- and the Z59-modified first-generation vectors in EGFRoverexpressing A431 carcinoma cells were increased in an antibody dose-dependent manner by up to 18-fold and 50-fold, respectively The increase in gene transfer levels was receptor-specific and could Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy be competitively blocked by protein A and by recombinant Zmodified knob protein, but not by wild-type knob By use of a Z59-modified helper virus, a high-capacity or ‘gutless’ vector was produced which could be targeted to EGFR in the same way Pretreatment of the Z33-modified first-generation vector with a monoclonal antibody recognizing integrin alpha7 considerably increased transduction of primary human smooth muscle and skeletal muscle cells In primary human myotubes, which are particularly refractory against Ad infection, preincubation of this vector with antibodies (at 40 microgram/ml) specific for the cell surface molecules integrin alpha7 or NCAM enhanced transduction efficiency by 68fold and 77-fold, respectively, as assessed by quantitative beta-Gal reporter gene assay The Ad vectors described hold great potential for targeting to a wide variety of cell types by simply exchanging the monoclonal antibody 413 Analysis of High-Capacity Adenovectors Expressing PEDF for Ocular Gene Therapy Joseph T Bruder,1 Ping Chen,1 Kurt Kroeger,1 Alena Lizonova,1 C R King.1 New Products Research, GenVec Inc., Gaithersburg, MD, United States Wet age-related macular degeneration (AMD) and diabetic retinopathy are the most common causes of blindness in the developed world Both diseases are caused by the abnormal growth of blood vessels in the eye Current treatments that destroy or inhibit the growth of existing blood vessels can delay vision loss but not affect the underlying disease and neovascularization is a recurring problem Pigment epithelium-derived factor (PEDF) is a potent anti-angiogenic and neuroprotective protein that is naturally produced in the eye When delivered to the eye via an adenovirus vector, PEDF can block growth of new blood vessels and trigger the regression of established abnormal vessels in animal models for AMD and diabetic retinopathy Two characteristics of AdPEDF that have been observed in animal models are the potential for vector induced inflammation and transient PEDF expression High-capacity (HC) adenovectors are characterized by the absence of viral coding sequence and are also referred to as helper-dependent, gutless or gutted vectors These vectors have been shown to be the least immunogenic, least toxic, and most persistent adenovectors to date Due to the absence of adenovirus genes, HC adenovectors offer the potential for persistent transgene expression and may greatly expand the therapeutic repertoire for adenovirus gene therapy vectors in the eye and other tissues We have built a functional system for producing gutless vectors that includes a cell line that expresses the FLPe recombinase (293FLPe17); a helper virus that has its packaging signal flanked by frt recombination sites; and HC vectors that express green fluorescent protein (GFP) and PEDF We observed efficient (>95% ) packaging signal excision at both 24 and 48hours after infection of 293FLPe17 cells with the frt helper virus In addition, helper virus yields were reduced by more than 500-fold in 293FLPe17 relative to 293 cells These results indicate that the FLPe system is functional We have used both this FLPe/frt and the Cre/Lox system to grow a HC vector that expresses the PEDF gene from the CMV promoter HC-vector preparations containing less than 1% helper contamination were obtained following CsCl purification We are currently investigating the inflammatory response to these vectors and the persistence of PEDF expression following delivery to the mouse eye I work for and hold stock in GenVec S163 ... resulted in a slightly greater percentage of recombination but also a decrease in cell viability In summary, we have constructed a HDA-EBV hybrid system capable of stably transforming mammalian cells. .. of AdPEDF that have been observed in animal models are the potential for vector induced inflammation and transient PEDF expression High- capacity (HC) adenovectors are characterized by the absence... first-generation vector with a monoclonal antibody recognizing integrin alpha7 considerably increased transduction of primary human smooth muscle and skeletal muscle cells In primary human myotubes,