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149. Transient Kupffer Cell Saturation, Via Adenovirus or Adenoviral Protein, Enhances Expression of Reporter Virus
ADENOVIRUS VECTORS: GENERAL BIOLOGY AND HOST RESPONSE and 64% and the Null vector produced elevations of 74%, 25% and 49% above the control at the same timepoints (p<0.01). CYP4A protein expression recovered by 14 days in each group. RT-PCR revealed that mRNA levels of CYP4A2, the primary CYP4A isoform responsible for renal fatty acid metabolism, increased by 16.8% and 19% above control 24 hours after administration of AdlacZ and Epo, respectively (p<0.01). CYP4F1, an isoform responsible for metabolism of fatty acids, prostaglandins and leukotrienes, was unaffected in all treatment groups. In contrast, renal CYP2E1, which metabolizes small lipophilic compounds and carcinogenic agents, was significantly reduced throughout the study. CYP2E1 mRNA levels in animals treated with AdlacZ were 74%, 78%, 69% and 66% of control at 0.25, 1, 4 and 14 days, respectively (p<0.01). Similar reductions were seen with Epo and Null vectors although levels form animals treated with Epo began to recover by 14 days. Real time quantitative PCR revealed that viral genomes (∼850/100 ng DNA) were initially present in the kidney and declined throughout the study for all treatments. These results suggest that systemic administration of Ad significantly altered the transcription and translation of several key CYP enzymes in the kidney regardless of the transgene expression cassette. This should be considered for accurate drug monitoring when employing recombinant Ad vectors in the clinic. Additional mechanistic studies assessing the effect of expression of Ad early genes and local and systemic eicosanoid biosynthesis on the expression of renal CYP will allow further explanation of these results.
149. Transient Kupffer Cell Saturation, Via Adenovirus or Adenoviral Protein, Enhances Expression of Reporter Virus 1
1
2
Viraj P. Mane, Christian Clarke, Lali Medina-Kauwe, Milton Finegold,1 Brendan Lee.1,3 1 Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 2Gene Therapeutics Research Institute, CedarsSinai Medical Center, Los Angeles, CA; 3Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX. Gene therapy vectors derived from adenoviral serotypes are efficacious both in vitro and in vivo, and are active in murine, canine, bovine and primate models. The development of gene-deleted HelperDependent Adenoviral vectors (HDAd) has attenuated chronic toxicity while improving transgene expression persistence. Nevertheless, early immune responses are raised against adenoviral vectors in a dose-dependent manner. Depending on dose, acute toxicity of the vector may culminate in a lethal proinflammatory condition in all model organisms including human. Kupffer cells are macrophages residing in the liver that have been implicated in the amplification of acute proinflammatory cascades in response to viral transduction. Their phogocytosis of adenovirus has also been implicated in a “threshold effect” whereby high viral doses follow a linear range of hepatic transduction secondary to Kupffer cell saturation, but low doses are mostly phagocytized and suffer highly restricted transgene expression. By immunofluorescence, we have confirmed that systemicallyadministered adenoviral vectors primarily localize to murine Kupffer cells. In addition, we found that systemic administration of an adenoviral predose (3x1012 viral particles/kg) one or eight hours before a low dose of reporter vector (1x1012 vp/kg) improves reporter transgene expression levels, corroborating previous reports. However, several murine toxicity measures were adversely impacted by administering adenovirus twice per animal, suggesting a cumulative toxic effect of adenovirus. Additionally, we explored the use of viral capsid protein predoses (purified fiber and penton) in an attempt to circumvent the aforementioned cumulative toxicities. Viral protein predoses were calculated as protein equivalencies to whole virus (i.e. one virion S60
corresponds to 12 trimeric fibers and 12 pentameric penton bases). Interestingly, we found that viral protein, at both intermediate (3x1012 vp/kg equivalency) and high (1x1013 vp/kg equivalency) predoses, did not exacerbate murine toxicities. The low toxicity of viral protein, however, was not accompanied by an enhancement of transgene expression. Finally, we administered viral protein simultaneously with reporter virus to assess toxicity and transgene activity. Both intermediate and high viral codoses had benign toxicity profiles. Interestingly, the high protein codose caused a doubling of reporter virus transgene activity. Current studies will address whether adenoviral proteins are capable of platelet activation, a condition for which adenoviral administration is a causative agent. Toxicities attributed to purified viral proteins will also be assayed. Furthermore, we will use immunofluorescence to determine whether adenoviral biodistribution is altered when viral proteins are co-administered with adenoviral doses. Our results suggest that a Kupffer cell saturation strategy may improve transgene expression from low doses of reporter virus without an adverse effect on toxicity.
150. Pre-Existing Nab and Neutralization of Therapeutic Dose Adenovirus Serotypes In Vitro Edward C. Nwanegbo,1 Wentao Gao,1 Andrea Gambotto,1 Paul Robbins.1 1 Surgery, Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA. Pre-existing Neutralizing antibody to adenoviral vectors is one of the limiting factors to the successful application of vectors in Gene Therapy. Studies have demonstrated a high and low prevalence of nab to Ad5 and Ad35 respectively in Africa, Asia, America, and Europe. While most report focused on the negative impact of this nab, a few have demonstrated successful gene transfer in the presence of nab. To further understand the Clinical implications of these findings, we studied the neutralization of Therapeutic and lower doses of the viruses using high and low concentration of pooled Human IgG and relate it to the existing titer of nab in the pooled Human IgG, Human Sera, and Sera from monkeys previously immunized and boosted with ad5 and ad35 Method: 1011viral particles and other doses, 1010,109,108 viral particles of Ad35 expressing Yellow Florescent protein, Ad5 expressing Green Florescent protein and Ad26 expressing Yellow Florescent Protein were incubated with 10mg, 7.5mg, 5mg, 2.5mg, and 0.5mg of IgG for 1hour at 37oC in 96 well plate. Freshly harvested A549 cells were seeded to the wells and incubated for 24hours at 37oC. For titer determination, serial dilutions of pooled Human IgG, Huaman and Monkey sera were incubated with 108 viral particles and treated in the same way. The plates were subsequently analyzed with Flourescent microscopy and by Flow cytometry. Result: High anti-Ad5 neutralizing antibody titer was associated with Neutralization of the therapeutic dose of the virus. Although low existing anti- Ad26 and Anti-Ad35 neutralizing antibody was observed, therapeutic doses of the viruses were not neutralized. Serum samples with high anti-Ad titer also neutralized 1011 viral particles of the particular serotype. Conclusion: Therapeutic and lower doses of Adenoviral vectors may be effectively used in Gene Therapy in the presence of preexisting low neutralizing antibody titer. While supra-therapeutic dose is required to overcome high Neutralizing antibody titer, a vector may be re-administered in the presence of low neutralizing antibody titer.
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
149. Transient Kupffer Cell Saturation, Via Adenovirus or Adenoviral Protein, Enhances Expression of Reporter Virus 1
1
2
Viraj P. Mane, Christian Clarke, Lali Medina-Kauwe, Milton Finegold,1 Brendan Lee.1,3 1 Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 2Gene Therapeutics Research Institute, CedarsSinai Medical Center, Los Angeles, CA; 3Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX. Gene therapy vectors derived from adenoviral serotypes are efficacious both in vitro and in vivo, and are active in murine, canine, bovine and primate models. The development of gene-deleted HelperDependent Adenoviral vectors (HDAd) has attenuated chronic toxicity while improving transgene expression persistence. Nevertheless, early immune responses are raised against adenoviral vectors in a dose-dependent manner. Depending on dose, acute toxicity of the vector may culminate in a lethal proinflammatory condition in all model organisms including human. Kupffer cells are macrophages residing in the liver that have been implicated in the amplification of acute proinflammatory cascades in response to viral transduction. Their phogocytosis of adenovirus has also been implicated in a “threshold effect” whereby high viral doses follow a linear range of hepatic transduction secondary to Kupffer cell saturation, but low doses are mostly phagocytized and suffer highly restricted transgene expression. By immunofluorescence, we have confirmed that systemicallyadministered adenoviral vectors primarily localize to murine Kupffer cells. In addition, we found that systemic administration of an adenoviral predose (3x1012 viral particles/kg) one or eight hours before a low dose of reporter vector (1x1012 vp/kg) improves reporter transgene expression levels, corroborating previous reports. However, several murine toxicity measures were adversely impacted by administering adenovirus twice per animal, suggesting a cumulative toxic effect of adenovirus. Additionally, we explored the use of viral capsid protein predoses (purified fiber and penton) in an attempt to circumvent the aforementioned cumulative toxicities. Viral protein predoses were calculated as protein equivalencies to whole virus (i.e. one virion S60
corresponds to 12 trimeric fibers and 12 pentameric penton bases). Interestingly, we found that viral protein, at both intermediate (3x1012 vp/kg equivalency) and high (1x1013 vp/kg equivalency) predoses, did not exacerbate murine toxicities. The low toxicity of viral protein, however, was not accompanied by an enhancement of transgene expression. Finally, we administered viral protein simultaneously with reporter virus to assess toxicity and transgene activity. Both intermediate and high viral codoses had benign toxicity profiles. Interestingly, the high protein codose caused a doubling of reporter virus transgene activity. Current studies will address whether adenoviral proteins are capable of platelet activation, a condition for which adenoviral administration is a causative agent. Toxicities attributed to purified viral proteins will also be assayed. Furthermore, we will use immunofluorescence to determine whether adenoviral biodistribution is altered when viral proteins are co-administered with adenoviral doses. Our results suggest that a Kupffer cell saturation strategy may improve transgene expression from low doses of reporter virus without an adverse effect on toxicity.
150. Pre-Existing Nab and Neutralization of Therapeutic Dose Adenovirus Serotypes In Vitro Edward C. Nwanegbo,1 Wentao Gao,1 Andrea Gambotto,1 Paul Robbins.1 1 Surgery, Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA. Pre-existing Neutralizing antibody to adenoviral vectors is one of the limiting factors to the successful application of vectors in Gene Therapy. Studies have demonstrated a high and low prevalence of nab to Ad5 and Ad35 respectively in Africa, Asia, America, and Europe. While most report focused on the negative impact of this nab, a few have demonstrated successful gene transfer in the presence of nab. To further understand the Clinical implications of these findings, we studied the neutralization of Therapeutic and lower doses of the viruses using high and low concentration of pooled Human IgG and relate it to the existing titer of nab in the pooled Human IgG, Human Sera, and Sera from monkeys previously immunized and boosted with ad5 and ad35 Method: 1011viral particles and other doses, 1010,109,108 viral particles of Ad35 expressing Yellow Florescent protein, Ad5 expressing Green Florescent protein and Ad26 expressing Yellow Florescent Protein were incubated with 10mg, 7.5mg, 5mg, 2.5mg, and 0.5mg of IgG for 1hour at 37oC in 96 well plate. Freshly harvested A549 cells were seeded to the wells and incubated for 24hours at 37oC. For titer determination, serial dilutions of pooled Human IgG, Huaman and Monkey sera were incubated with 108 viral particles and treated in the same way. The plates were subsequently analyzed with Flourescent microscopy and by Flow cytometry. Result: High anti-Ad5 neutralizing antibody titer was associated with Neutralization of the therapeutic dose of the virus. Although low existing anti- Ad26 and Anti-Ad35 neutralizing antibody was observed, therapeutic doses of the viruses were not neutralized. Serum samples with high anti-Ad titer also neutralized 1011 viral particles of the particular serotype. Conclusion: Therapeutic and lower doses of Adenoviral vectors may be effectively used in Gene Therapy in the presence of preexisting low neutralizing antibody titer. While supra-therapeutic dose is required to overcome high Neutralizing antibody titer, a vector may be re-administered in the presence of low neutralizing antibody titer.
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy