Readministration Of Vector Research Articles

Does the use of recombinant AAV5 in pulmonary gene therapy lead to lung damage?

This study investigated whether repeated administration of recombinant adeno-associated virus type 5 (rAAV5) to the airways induces inflammatory processes in the lungs of BALB/c-mice, with mechanical and histologic changes. Saline was instilled intratracheally in the control group, and rAAV5-green fluorescence protein (GFP) (4x10(11)particles) in the virus group (VR). These groups were subdivided into four subgroups: one dose analyzed 3 weeks later (VR1d3w) and two doses analyzed 1 (VR2d1w), 2 (VR2d2w) and 3 weeks (VR2d3w) after the second dose. Lung morphometry, mechanical parameters, airway responsiveness, rAAV5-GFP transduction and the expression of inflammatory cytokines were investigated. No significant differences in lung mechanics, airway responsiveness, and morphometry were observed. Re-administration of rAAV5 vector resulted in a decrease in GFP mRNA expression in the VR2d3w group. There was no evidence of inflammatory response or apoptosis in any group. rAAV5 did not induce an inflammatory process, mechanical or morphometric changes in the lungs. AAV5 may be an appropriate vector for lung gene therapy.

Transient Pretreatment With Glucocorticoid Ablates Innate Toxicity of Systemically Delivered Adenoviral Vectors Without Reducing Efficacy

More than 300 human clinical trials utilize recombinant adenoviruses (rAds) as a gene transfer vector, confirming that rAds continue to be of high clinical interest. A primary weakness of rAds is their known propensity to trigger an innate, proinflammatory immune response rapidly after high-dose, systemic administration. In this study, we investigated what affects that pre-emptive treatment with anti-inflammatory glucocorticoids might have upon Ad vector-triggered inflammatory immune responses. We found that a simple pretreatment regimen with Dexamethasone (DEX) can significantly reduce most Ad-induced innate immune responses. DEX prevented rAd induction of systemic cytokine/chemokine releases in a dose-dependent fashion, with higher dosages preventing rAd induction of acute thrombocytopenia, endothelial cell activation, proinflammatory gene induction, and leukocyte infiltration into transduced organs. Transient glucocorticoid pretreatment also significantly reduced rAd-induced adaptive immune responses, including a decreased induction of Ad-neutralizing antibodies (NAbs). Importantly, use of DEX did not reduce the efficacy of rAd-mediated gene transduction nor rAd-derived transgene expression. Our results demonstrate that a simple, pre-emptive and transient glucocorticoid pretreatment is a viable approach to reduce rAd-associated acute toxicities that currently limit the use of Ad vectors in systemic clinical applications.

Coating of AAV Vectors with Human Albumin Blocks Antibody Binding and Enables Transduction of Human Hepatocytes in the Presence of Neutralizing Antibodies

Neutralization of recombinant adeno-associated virus (AAV) gene transfer vectors by pre-existing antibodies is a significant barrier to clinical gene transfer using systemic administration. In a recent clinical trial for hemophilia B, while efficient gene transfer and therapeutic levels of hFIX were achieved in a human subject with low pre-existing antibodies to AAV2, no gene transfer was observed in a subject with a modest pre-existing AAV2 antibody titer of 1:17 who received the same dose (Manno et al. Nature Med. 2006; 12:342–347). With the objective to achieve consistent and efficient transduction in the presence of AAV antibodies, ‘humanized’ AAV vectors were generated by covalent attachment of human albumin (HSA) to the surface of purified AAV2 by cross linking the single free thiol (Cys34) of HSA to lysine residues on the vector surface using suflosuccinimidyl 4 [N-maleimidomethyl] cyclohexane-1-caboxylate (Sulfo-SMCC). Variable levels of HSA substitution up to 65 HSA molecules per virion were achieved, corresponding to a maximum of approximately one HSA molecule per virion capsid protein. Analysis by SDS-PAGE of the modified vector revealed the presence of a 130kDA polypeptide species in the conjugated vector, not observed in the unmodified vector, that was consistent with predicted molecular weight obtained by covalent attachment of HSA with AAV capsid protein VP3. Dynamic light scattering demonstrated an average radius of 16.6 nm for the HSA-conjugated vector compared with 11.6 nm for unconjugated vector, consistent with coating of the vector surface with HSA. Analysis by flow cytometry demonstrated that the binding of AAV2-specific monoclonal antibody A20 to unmodified vectors was progressively reduced to background levels in HSA-conjugated vectors with increasing HSA substitution. Quantitative analysis by Biacore indicated that the association rate and available sites per virion for antibody binding were reduced > 10- fold in HSA-conjugated vectors. The conjugated vectors were significantly more resistant than unmodified vectors to neutralization by AAV antibody, demonstrating comparable transduction in the presence of 16-fold higher concentration of MAb A20 following transduction of cultured human hepatocytes (HepG2). In conclusion, covalent coupling of HSA to the surface of recombinant AAV2 is demonstrated as a feasible method to modify AAV vectors to increase their resistance to neutralizing antibodies. ‘Humanized’ AAV vectors such as HSA-coated AAV that effectively mask viral epitopes recognized by antibodies and display human epitopes may enable more consistent transduction following systemic administration in vivo, and enable vector readministration.

DNA Shuffling of Adeno-associated Virus Yields Functionally Diverse Viral Progeny

Adeno-associated virus (AAV) vectors are extremely effective gene-delivery vehicles for a broad range of applications. However, the therapeutic efficacy of these and other vectors is currently limited by barriers to safe, efficient gene delivery, including pre-existing antiviral immunity, and infection of off-target cells. Recently, we have implemented directed evolution of AAV, involving the generation of randomly mutagenized viral libraries based on serotype 2 and high-throughput selection, to engineer enhanced viral vectors. Here, we significantly extend this capability by performing high-efficiency in vitro recombination to create a large (10(7)), diverse library of random chimeras of numerous parent AAV serotypes (AAV1, 2, 4-6, 8, and 9). In order to analyze the extent to which such highly chimeric viruses can be viable, we selected the library for efficient viral packaging and infection, and successfully recovered numerous novel chimeras. These new viruses exhibited a broad range of cell tropism both in vitro and in vivo and enhanced resistance to human intravenous immunoglobulin (IVIG), highlighting numerous functional differences between these chimeras and their parent serotypes. Thus, directed evolution can potentially yield unlimited numbers of new AAV variants with novel gene-delivery properties, and subsequent analysis of these variants can further extend basic knowledge of AAV biology.

Gene Delivery to the Juvenile Mouse Liver Using AAV2/8 Vectors

Recombinant adeno-associated viral (rAAV) vectors have shown promise for use in liver-targeted gene delivery, but their effects have not been extensively investigated in the immature liver. Understanding the impact of liver growth on the efficacy of transduction is essential, because many monogenic liver diseases that are amenable to gene therapy will require treatment early in life. Here we show that rAAV2/8 transduces the neonatal mouse liver with high efficiency. With just one doubling in liver weight, however, there is a rapid reduction in vector genome numbers, irrespective of form, and the loss of episomal vector is almost complete by 2 weeks. Stable transgene expression is observed in a small percentage of hepatocytes, often in two- to eight-cell clusters, suggestive of genomic integration. Delivery at serially older ages was associated with progressively improved episome persistence and transgene expression. Vector re-administration was possible following initial neonatal administration, albeit at reduced efficacy because of an anticapsid humoral immune response. We also found that intraperitoneal (IP) delivery of rAAV2/8 was highly effective at all ages, and that promoter selection is the critical determinant of the intensity and pattern of transgene expression across the hepatic lobule. We conclude that successful use of rAAV to treat liver disease in early childhood will require optimally efficient vector constructs and probable re-administration.

903. Pulmonary Transduction in Nonhuman Primates by HDAd: Duration of Transgene Expression and Vector Re-Administration

903. Pulmonary Transduction in Nonhuman Primates by HDAd: Duration of Transgene Expression and Vector Re-Administration

Prolonged Susceptibility to Antibody-mediated Neutralization for Adeno-associated Vectors Targeted to the Liver

Adeno-associated virus (AAV) vectors demonstrate highly efficient gene transfer to hepatocytes in vivo. One of the remaining obstacles to the treatment of hemophilia B patients with AAV vectors is the sensitivity of these vectors to antibody-mediated neutralization following systemic delivery. Testing and implementation of strategies to circumvent pre-existing antibodies requires knowledge of the clearance kinetics of AAV from circulation. In this study, AAV clearance kinetics were established for serotypes 2 and 8 in cell culture and in mice. Administration of pooled neutralizing serum subsequent to administration of the vector was used to define the time period in which the vector is susceptible to antibody-mediated neutralization. These experiments defined the in vivo clearance rates for both AAV2 and AAV8 vectors to be between 2 and 4 hours. In mice, portal vein and tail vein administration of each vector was tested with similar results. Cell culture studies in W162 cells established that cellular attachment and internalization both contribute to the clearance kinetics of AAV vectors. These studies characterize the in vivo clearance rates of AAV vectors for the first time and guide the development of future strategies for the avoidance of antibody-mediated AAV vector neutralization.

Effect of viral dose on neutralizing antibody response and transgene expression after AAV1 vector re-administration in mice

Neutralizing antibodies (nAB) at the time of administration hamper the effectiveness of adeno-associated virus (AAV) as a clinical DNA delivery system. The present study was designed to investigate if AAV re-administration in muscle tissue is dependent on the nAB titer. Recombinant (r)AAV serotype 1, as a promising candidate for targeting skeletal muscle, was used for gene delivery. C57Bl/6 mice were infected intramuscularly with doses between 1 x 10(9) and 5 x 10(10) virus particles (vp) of AAV1-expressing luciferase (AAV1-luc) or human interferon-beta (AAV1-hIFNbeta). Increasing transgene expression was observed over the first 2 months and anti-AAV1 nAB titers peaked between weeks 4 and 8. Six months after the first administration, 5 x 10(10) vp of AAV1-IFNbeta were re-administered. Following re-administration, nAB titers increased but did not significantly affect transgene expression from the AAV vector that had been administered first. In contrast, hIFNbeta expression originating from the second vector administration was significantly diminished and reflected the nAB titer present at the day of re-administration. The present study extends earlier observations that preexisting nAB affects AAV1 re-administration. The level of nAB is proportional to the virus dose used for the first injection and transgene expression following re-administration is dependent on preexisting nAB titer.

Readministration of adenoviral gene delivery to dopamine neurons

An approach currently being explored as treatment for Parkinson's disease is gene therapy. An important question concerns the duration of transgene expression in dopamine neurons and the issues of vector persistence, neuronal damage and the feasibility of readministering vector to the same neuronal population. We show, using an adenoviral vector expressing the LacZ reporter gene, that transgene expression declined over time but with minimal loss of dopamine neurons or vector DNA. Readministration of vector resulted in low levels of transgene delivery to the neurons. Moreover, the neurons to which vector had already been delivered were unable to transport the retrograde tracer fluorogold. Our findings indicate that transgene expression declined in dopamine neurons despite the persistence of virus, and the capacity to readminister vector to these neurons was limited.

Pulmonary delivery of adenovirus vector formulated with dexamethasone–spermine facilitates homologous vector re-administration

Gene transfer to lung has been hindered by inflammatory and immunological responses activated to the gene-transfer agent or transgene products. In prior work, adenovirus vector delivered to the lung with the cationic glucocorticoid, dexamethasone-spermine (DS) had improved targeting to conducting airway epithelium and reduced cellular infiltration. In this study, the effect of formulation on homologous adenovirus vector re-administration was studied in C57Bl/6 mice. Formulation of an adenovirus vector expressing LacZ with DS/dioleoylphosphatidylethanolamine (DOPE) delivered at day 0 allowed re-administration of adenovirus vector expressing alkaline phosphatase at day 21. Formulation with 3beta [N-(N', N'-dimethylaminoethane) carbamoy] cholesterol (DC-Chol) DC-cholesterol (DC-Chol))/DOPE or dexamethasone in the first dosing at day 0 resulted in moderate alkaline phosphatase expression at day 24. Neutralizing antibodies against adenovirus vector in serum at day 28 were greatly reduced by all three formulations in mice receiving a single dose of adenovirus at day 0. Also, homologous adenovirus vector re-administration at day 14 produced less neutralizing antibody at day 28 when adenovirus was formulated with DS/DOPE at day 0. The use of DS/DOPE at day 0 dramatically reduced CD4 and CD8 T-cell infiltration in mice receiving adenovirus at day 0 followed by vector re-administration at day 14. Transgene-specific T-cell activation was markedly reduced by the DC-Chol/DOPE formulation. Overall, DS/DOPE) facilitated homologous vector re-administration through a combination of liposomal and glucocorticoid mechanisms.

Lack of Repeat Transduction by Recombinant Adeno-Associated Virus Type 5/5 Vectors in the Mouse Airway

While recombinant adeno-associated virus (rAAV) vectors promote long-term transgene expression in the lungs and other organs, the goal of correcting chronic inherited lung diseases such as cystic fibrosis with this type of viral gene transfer vector is limited by the requirement of achieving stable potent transgene expression, potentially requiring vector readministration. Here we evaluated the abilities of rAAV type 5/5 (rAAV5/5) vectors based on the genome and capsid of AAV5 to efficiently transduce the lungs and nasal epithelium of mice after repeated administration. Transduction efficiency as judged by reporter gene expression was markedly reduced on a second rAAV5/5 administration and effectively abolished on a third. Varying the period between administrations from 8 to 36 weeks did not allow efficient repeated administration. A rapid rise in anti-AAV5 antibodies was noted after rAAV5/5 vector administration that was sustained for the entire period of investigation (in some cases exceeding 9 months). Furthermore, this antibody response and subsequent failure to repeatedly administer the vector were not rescued by the in vivo expression of CTLA4Ig from an rAAV5/5 vector. These results suggest that without the development of an effective and clinically acceptable immunosuppression strategy, treatments for chronic diseases that require repeated administration of rAAV5/5 vectors will be unsuccessful.

903. Re-Administration of Recombinant AAV2 and AAV5 in the Brain

Adeno-associated virus has been extensively examined as a vector for gene delivery. Unlike adenovirus or herpes virus administration, there is little immune response systemically, and within the central nervous system the immune response is negligible. rAAV2 has been the prototype vector and is the most common vector studied. It has proven to be an efficient and reliable vector, however as much as 80% of the human population has antibodies to this serotype, with many having neutralizing antibodies. The AAV2 vector uncoats slowly and the presence of the capsid may lead to an immune response. Previous research has examined administration of rAAV2 after exposure to wild-type AAV2 or rAAV2. These studies revealed that re-administration of rAAV2 results in a decrease (or abolition) of transduced neurons by rAAV2. Many new serotypes of AAV have been discovered and examined in the past few years. It has been shown that AAV5 uncoats faster than AAV2 and this may make it possible for the vector to escape a prolonged exposure to the immune system, making it less likely that the immune system will be primed. Alternately, GFP is also immunogenic and re-administration of rAAV2 carrying GDNF does not result in decreased transduction, revealing that immune response may depend on the gene product being produced. Therefore it is important to determine whether the capsid is triggering an immune response or is GFP the immunogen. This study was designed to examine the number of striatal neurons transduced after administration of rAAV2-GFP or rAAV5-GFP and re-administration of either vector. Preliminary results indicate that re-administration of the rAAV5 vector does not lead to loss of transduction and may be a viable option when re-administration is necessary. Preliminary results indicate that the immune response is directed at the capsid, and not the GFP.

785. A Novel Adeno Associated Virus Vector That Efficiently Transduces Airway Epithelium In Vivo and Can Be Re-Administered

Top of pageAbstract Airway|[ndash]|directed gene transfer has emerged as a promising approach for the treatment of cystic fibrosis and |[alpha]|-1-antitrypsin deficiency (AAT). It will be important to re-administer the vector for treatment of these chronic disorders. We have shown previously that AAV2/9 can efficiently target and transduce the murine upper and lower airways in vivo. As such we studied the performance of AAV2/9 in the setting of pre-existing immunity to homologous vector. C57Bl/6 mice were inoculated intratracheally with a single dose (1E+11 genome copies) of either AAV2/9 or AAV2/5 expressing AAT. We found that AAV2/9-mediated AAT expression was relatively stable for the duration of the experiment (9 months) and at the peak of gene expression was 60-fold higher than that of AAV2/5. Within two weeks of vector administration, high levels (>1:2560) of serum|[ndash]|circulating neutralizing antibodies (NAB) were observed in both AAV2/9 and AAV2/5 treated mice. Mice were divided into groups and designated groups were subjected to a second instillation of the same vector serotype expressing a different transgene (nLacZ) at specific time points after delivery of the AAV2/9- or AAV2/5|[ndash]| AAT vectors. Mice were sacrificed 21 days following the AAV|[ndash]| nLacZ vector instillation and the lung and nose processed for nLacZ expression. The serum NAB titers in the AAV2/9 treatment group remained high (average of 1:2560) up to day 112 and decreased to |[sim]|1:640 by day 217. Interestingly, we found that AAV2/9 was able to circumvent the AAV2/9-specific NABs, resulting in nLacZ gene transfer levels in nasal and lung airways similar to that of age-matched naive mice at all time points. In AAV2/5-treated mice, no transduced cells were seen in either the nasal or lung airways over a broad range of serum|[ndash]|circulating NAB titers (1:56000 to 1:1280). Some level of gene transfer was achieved with AAV2/5-nLacZ when administered 196 days after the AAT vector which was when serum NAB declined to 1:320|[ndash]|1:160. To explain why AAV2/9 can efficiently transduce the lung airway epithelium in the presence of high titer serum|[ndash]| circulating NAB, the bronchoalveolar lavage fluid (BALF) isolated from mice at the first time point (day 49) and at the end of the experiment (day 217) was analyzed for AAV-specific NABs. We did not observe any NAB titer, IgG or IgA responses to AAV2/9 vector. In contrast, when BALF from AAV2/5 treated mice was analyzed, NAB titers ranged from 1:160 to 1:1280, vector IgG responses from 1:100 to 1:12800 and vector IgA responses from 1:100|[ndash]|1:1600. Our results suggest that AAV2/9 is an excellent candidate for clinical applications that require long term transgene expression. We show that AAV2/9 can be re-administered to lung with out diminution of gene transfer efficiency while AAV2/5 can not. This finding further supports the development of AAV2/9 for use in clinical applications for chronic diseases in which vector re-administration will be necessary.

983. Repeat Intra-Articular Administration of AAV2 Vectors to Rat Joints

Rheumatoid arthritis is a chronic autoimmune disease characterized by synovial inflammation, leading to the destruction of cartilage and bone. Previously, we demonstrated that a single intra-articular administration of AAV2-TNFR:Fc vector to rats with experimental arthritis resulted in suppression of disease. For treatment of chronic inflammatory arthritis using intra-articular delivery of AAV2- TNFR:Fc vector, intra-articular re-administrations may be required. In this study, we evaluated if serum anti-AAV2 capsid neutralizing antibodies, elicited after intra-articular administration of AAV2- TNFR:Fc vector to rat joints, affect joint transduction following intra-articular re-administration of an AAV2-FlagLuc vector.

988. Repeat Administration of Lentiviral Vector to Mouse Nasal Epithelia

Adverse immune responses to vector-encoded proteins may hinder the practical application of gene therapy. To date, little effort has been devoted to determining the immunological consequences of topical delivery of lentiviral vectors to respiratory epithelia. Previously, we demonstrated that an integrating feline immunodeficiency virus-based lentiviral vector pseudotyped with a baculoviral envelope glycoprotein (GP64-FIV) readily transduced differentiated airway epithelia in vitro when applied to the apical surface. Furthermore, using a luciferase reporter gene and bioluminescence imaging, we observed in vivo gene transfer to the mouse nasal epithelia following a single application of GP64-FIV. Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for greater than 11 months without significant decline. By histological analysis, surface epithelial cells were transduced following a single nasal application of GP64-FIV expressing beta-galactosidase. Importantly, we observed that 7 consecutive doses of GP64-FIV delivered over 7 consecutive days led to greatly increased number of beta-galactosidase positive epithelial cells. However, using quantitative bioluminescent imaging, we observed that repeated doses given over consecutive days did not result in a linear increase in gene expression. We have now initiated studies investigating gene transfer efficacy and immune responses following lentiviral vector readministration with greater intervening time periods. In control mice, we observed that giving 2 nasal instilled doses (2 weeks apart) of an E1/E3 deleted Ad5 vector resulted in significantly attenuated expression of the second dose. In contrast, using the same delivery protocol, 2 doses of GP64-FIV did not result in loss of expression of the second dose. Ongoing studies are investigating the impact of lentiviral vector readministration on marker gene expression, as well as how gene transfer efficiency correlates with the production of neutralizing antibodies. GP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia and has the potential for repeat administration without eliciting adverse immune responses. These data have important implications for lentiviral gene therapy.

995. Effect of Viral Dose on Neutralizing Antibody Response and Transgene Expression after AAV1 Vector Re-Administration in Mice

Pre-existing neutralising antibodies (NAB) at the time of administration hamper the effectiveness of adeno-associated virus (AAV) as a DNA delivery system in humans. Several studies demonstrated that there is a high prevalence of NAB directed against AAV2 in human sera limiting the efficacy of first administration. Recent studies have shown that there are no or very low levels of pre-existing NAB for AAV1. However, the development of NAB might become a hurdle when a second AAV1 administration is necessary.

Utility of PEGylated recombinant adeno-associated viruses for gene transfer

Adeno-associated virus (AAV), capable of producing significant, long-term transgene expression, is one of the least toxic vectors employed in pre-clinical and clinical studies of gene transfer. One limitation is generation of neutralizing antibodies against viral capsids, blocking gene expression after readministration. AAV2 capsids were modified with poly(ethylene) glycols (PEGs) activated by cyanuric chloride (CCPEG), succinimidyl succinate (SSPEG) and tresyl chloride (TMPEG). SSPEG and TMPEG conjugation did not compromise gene transfer to the liver and muscle and improved gene expression in the lung 5 fold. Transduction efficiency of CCPEG-AAV was impeded in all tissues by aggregation. TMPEG afforded the best protection from neutralization in vitro and in vivo. Gene expression in mice immunized against unmodified AAV was reduced by a factor of 10 from that of naïve animals after intramuscular rechallenge with PEGylated AAV but was not significantly different from naïve mice after intravenous readministration ( p = 0.08). Gene expression was markedly reduced in muscle after two doses of PEGylated AAV. In contrast, mice given two intravenous doses of TMPEG-AAV had significantly higher transgene levels than naïve animals 14 days after rechallenge ( p = 0.001). This technology could promote successful readministration of vector in the clinic and marked expression in patients with anti-AAV antibodies.

Immune Responses to Adeno-Associated Virus Vectors

One of the biggest challenges in optimizing viral vectors for gene therapy relates to the immune response of the host. Adeno-associated virus (AAV) vectors are associated with low immunogenicity and toxicity, resulting in vector persistence and long-term transgene expression. The inability of AAV vectors to efficiently transduce or activate antigen presenting cells (APCs) may account for their decreased immunogenicity. AAV mediated gene therapy however, leads to the development of antibodies against the vector capsid. Anti-AAV antibodies have neutralizing effects that decrease the efficiency of in vivo gene therapy and can prevent vector re-administration. Furthermore, recent studies have shown that AAV vectors can elicit both cellular and humoral immune responses against the transgene product. Both cell-mediated response and humoral response to the delivered gene depend on a number of variables; including the nature of the transgene, the promoter used, the route and site of administration, vector dose and host factors. The response of the host to the vector, in terms of antigen-specific immunity, will play a substantial role in clinical outcome. It is therefore important to understand both, why AAV vectors are able to escape immunity and the circumstances and mechanisms that lead to the induction of immune responses. This review will summarize innate and adaptive immune responses to AAV vectors, discuss possible mechanisms and outline strategies, such as capsid modifications, use of alternative serotypes, or immunosuppression, which have been used to circumvent them.

Use of chimeric adenoviral vectors to assess capsid neutralization determinants

In order to elucidate the relative importance of neutralizing determinants on each of the three major adenoviral capsid components, we have generated chimeric vectors where the hexon protein, or the fiber protein, or both hexon and fiber proteins of one serotype (Simian Adenovirus 24/Pan 7) have been replaced by those of another (Simian Adenovirus 23/Pan 6). The effect of each replacement was evaluated by neutralization assays and by attempted vector re-administration into mice. Both hexon and fiber were found to harbor neutralization epitopes although in vivo transduction was more severely affected by anti-hexon antibodies.

127. Adenovirus Type 5 Vectors Pseudotyped with Fibers from Subgroup D (19p and 37) Show Detargeting from Hepatocytes In Vitro and In Vivo

Use of adenoviral gene therapy vectors based on different non-cross reacting serotypes is being investigated by several groups as a means to administer adenoviral vectors, either in a re-administration setting, or in subjects with circulating antibodies against the common serotypes such as Ad2 or Ad5. We have previously developed GFP expressing vectors using the chimpanzee adenoviruses Pan 6 (C6-GFP) and Pan 7 (C7-GFP). We have determined that administration of either one of the two chimpanzee adenovirus vectors does not affect the transduction efficiency of the other vector while re-administration of the same vector is severely compromised. Adenoviruses have three major capsid proteins, hexon, penton base, and fiber, which elicit the production of circulating antibodies. We wished to elucidate the relative importance of each of the three capsid components in preventing transduction in a gene therapy setting. In order to do this we have generated a panel of GFP expressing vectors that are chimeric between Pan 6 and Pan 7 where the hexon protein or the fiber protein or both hexon and fiber proteins of the Pan 7 vector have been replaced by those from Pan 6. Balb/C mice were immunized with either Pan 6 or Pan 7 and re-administration (by tail vein injection) was attempted 3 weeks later using each of the five GFP expressing vectors (C6-GFP, C7-GFP, and the three chimeric vectors). Three days later the level of liver transduction was estimated qualitatively by examining liver sections for the presence of GFP expression and quantitatively by estimating copies of GFP DNA by Taqman analysis. The data showed that antibodies to both hexon and fiber are important in preventing re-administration of adenoviral vectors.