Liver Transduction Efficiency Research Articles

Molecular Analysis of Vector Genome Structures After Liver Transduction by Conventional and Self-Complementary Adeno-Associated Viral Serotype Vectors in Murine and Nonhuman Primate Models

Vectors based on several new adeno-associated viral (AAV) serotypes demonstrated strong hepatocyte tropism and transduction efficiency in both small- and large-animal models for liver-directed gene transfer. Efficiency of liver transduction by AAV vectors can be further improved in both murine and nonhuman primate (NHP) animals when the vector genomes are packaged in a self-complementary (sc) format. In an attempt to understand potential molecular mechanism(s) responsible for enhanced transduction efficiency of the sc vector in liver, we performed extensive molecular studies of genome structures of conventional single-stranded (ss) and sc AAV vectors from liver after AAV gene transfer in both mice and NHPs. These included treatment with exonucleases with specific substrate preferences, single-cutter restriction enzyme digestion and polarity-specific hybridization-based vector genome mapping, and bacteriophage phi29 DNA polymerase-mediated and double-stranded circular template-specific rescue of persisted circular genomes. In mouse liver, vector genomes of both genome formats seemed to persist primarily as episomal circular forms, but sc vectors converted into circular forms more rapidly and efficiently. However, the overall differences in vector genome abundance and structure in the liver between ss and sc vectors could not account for the remarkable differences in transduction. Molecular structures of persistent genomes of both ss and sc vectors were significantly more heterogeneous in macaque liver, with noticeable structural rearrangements that warrant further characterizations.

Optimized Adeno-Associated Virus (AAV)–Protein Phosphatase-5 Helper Viruses for Efficient Liver Transduction by Single-Stranded AAV Vectors: Therapeutic Expression of Factor IX at Reduced Vector Doses

Abstract Our studies have shown that coinjection of conventional single-stranded adeno-associated virus 2 (ssAAV2) vectors carrying the enhanced green fluorescent protein (EGFP) gene with self-complementary (sc) AAV2-T cell protein tyrosine phosphatase (TC-PTP) and scAAV2-protein phosphatase-5 (PP5) vectors resulted in an approximately 16-fold increase in EGFP expression in primary murine hepatocytes in vivo [Jayandharan, G.R., Zhong, L., Li, B., Kachniarz, B., and Srivastava, A. (2008). Gene Ther. 15, 1287-1293]. In the present studies, this strategy was further optimized to achieve transgene expression at reduced vector/helper virus doses. These included the use of scAAV helper viruses containing (1) hepatocyte-specific promoters, (2) tyrosine-mutant AAV2 capsids, and (3) additional AAV serotype vectors known to efficiently transduce hepatocytes. The hepatocyte-specific transthyretin (TTR) promoter was approximately 6- to 7-fold more efficient than the Rous sarcoma virus (RSV) promoter; tyrosine-mutant AAV2 capsids were approximately 6- to 11-fold more efficient than the wild-type AAV2 capsids; and the AAV8 serotype helper virus was approximately 16-fold more efficient than AAV2 serotype helper virus. With these modifications, the vector dose of the helper virus could be further reduced by approximately 50-fold. Last, coadministration of scAAV8-PP5 helper virus increased coagulation factor IX expression from an ssAAV2 vector by approximately 7- to 10-fold, thereby achieving therapeutic levels at lower vector doses. No adverse effect on hepatocytes was observed under any of these experimental conditions. The strategy presented here should be adaptable to any ssAAV transgene cassette and, specifically, liver-directed applications of ssAAV2 vectors containing larger genes that cannot be encapsidated in scAAV vectors.

Effect of Adeno-Associated Virus Serotype and Genomic Structure on Liver Transduction and Biodistribution in Mice of Both Genders

Recombinant adeno-associated viral (AAV) vectors have unique properties, which make them suitable vectors for gene transfer. Here we assess the liver transduction efficiency and biodistribution of AAV-pseudotyped capsids (serotypes) 1, 5, 6, and 8, combined with single-stranded and double-stranded genomic AAV2 structures carrying the luciferase reporter gene after systemic administration. The analysis was performed in vivo and ex vivo, in male and female mice. Gender-related differences in AAV-mediated transduction and biodistribution were shown for the four serotypes. Our data confirm the superiority of AAV8 over the rest of the serotypes, as well as a significant advantage of double-stranded genomes in terms of liver transduction efficiency, particularly in females. Regarding biodistribution, AAV5 displayed a narrower tropism than the other serotypes tested, transducing, almost exclusively, the liver. Interestingly, AAV1 and AAV8, in particular those having single-stranded genomes, showed high transduction efficiency of female gonads. However, no inadvertent germ line transmission of AAV genomes was observed after breeding single-stranded AAV8-injected female mice with untreated males. In conclusion, double-stranded AAV8 vectors led to the highest levels of liver transduction in mice, as demonstrated by luciferase expression. Nevertheless, the transduction of other organs with AAV8 vectors could favor the use of less efficient serotypes, such as AAV5, which display a narrow tropism.

Comparative Study of Anti-hepatitis B Virus RNA Interference by Double-stranded Adeno-associated Virus Serotypes 7, 8, and 9

Using a hepatitis B virus (HBV) transgenic mouse model, we previously showed that a single dose of double-stranded adeno-associated virus (dsAAV) vector serotype 8 carrying a small hairpin RNA (shRNA) effectively reduces HBV replication and gene expression, but the effect gradually decreases with time. In this report, we compared the anti-HBV RNA interference (RNAi) effect of dsAAV8 with those of dsAAV7 and dsAAV9, two other hepatotropic AAV vectors, and examined whether the sequential use of these heterologous AAV vectors could prolong the anti-HBV effect. Our results showed that shRNA delivered by each of the three dsAAV vectors profoundly reduced the serum HBV titer and liver HBV mRNA and DNA levels in the transgenic mice for up to 22 weeks, with dsAAV8 having the greatest inhibitory effect, followed by dsAAV9 and dsAAV7. The potency of dsAAV8 correlated with the presence of higher levels of vector DNA and anti-HBV shRNA in the liver. An in vivo cross-administration experiment showed that preexisting anti-AAV8 antibody completely blocked the anti-HBV RNAi effect of dsAAV8, but had no effect on the potency of dsAAV7 and dsAAV9. Moreover, we demonstrated that a longer anti-HBV effect could be achieved by the sequential use of dsAAV8 and dsAAV9. These results indicate that effective and persistent HBV suppression might be achieved by a combination of the power of RNAi silencing effect and multiple treatments with different AAV serotypes.Molecular Therapy (2009) 17 2, 352-359 doi:10.1038/mt.2008.245.

Biochemical, Pathological, and Skeletal Improvement of Mucopolysaccharidosis VI After Gene Transfer to Liver but Not to Muscle

Mucopolysaccharidosis VI (MPS VI) is caused by deficient activity of arylsulfatase B (ARSB), resulting in intralysosomal storage of dermatan sulfate (DS) and multisystem disease without central nervous system involvement. After gene transfer, muscle or liver can theoretically be converted into factories for systemic ARSB secretion, leading to uptake by non-transduced cells. We have injected newborn MPS VI rats and cats with adeno-associated viral (AAV) vectors expressing ARSB under the control of liver-specific, muscle-specific, or universally active promoters. After systemic or intramuscular (IM) administration of AAV, therapeutic levels of circulating ARSB are achieved, resulting in skeletal improvements and significant decrease in glycosaminoglycan (GAG) storage, inflammation and apoptosis (despite a neutralizing immune response to ARSB in MPS VI rats). In addition, we have observed wide-spread dissemination of vector after IM AAV administration. This results in secretion of therapeutic levels of ARSB when the universally active cytomegalovirus (CMV) but not the muscle-specific muscle creatine kinase (MCK) promoter is used, suggesting that transduction of extramuscular sites rather than enzyme secretion from muscle occurs after muscle ARSB gene transfer. We conclude that AAV-mediated expression of ARSB from liver represents a feasible therapeutic strategy for MPS VI, potentially avoiding multiple infusions of costly recombinant enzyme associated with enzyme replacement therapy.

Safety of Recombinant Adeno-Associated Viral Vectors in a Large Animal Model.

Recombinant adeno-associated viral (AAV) vector is a promising gene-based strategy for the treatment of several inherited diseases. Using AAV serotype 2 (AAV-2), the most common tested vector in humans, we have determined that the risk of germline transmission and the immune responses to both transgene product and/or vector-capsid proteins are critical obstacles to the safety of this strategy (Nat Med 12:342, 2006). Recently, novel and more potent serotypes have emerged such as AAV-8 that allows efficient liver transduction following peripheral intravenous injection (IV). The major determinant of vector safety relies on its tissue tropism. Here, we sought to compare the efficacy and safety profile of AAV-2 and AAV-8 in a large animal model. Male rabbits (∼3 kg) received IV injection of AAV-2 (n=11) or AAV-8 (n=11) encoding human F.IX (hF.IX) at doses ranging from 1×1012 to 1×1013vg/kg. Injections with AAV-2-hF.IX resulted in 6-fold lower expression of hF.IX than AAV-8-hF.IX for both low and high dose cohorts. Notably, no neutralizing antibody to hF.IX was detected with either serotypes. Eighteen weeks following the initial injections, animals were cross-administered with either AAV-2 or AAV-8. Whereas injection of AAV-8 led a 20% increase in transgene expression in animals initially injected with AAV-2-h.FIX, AAV-2 failed to boost hF.IX expression. Regarding germline safety, the presence of vector genomes in semen samples from the high-dose cohort (6 to 10 weeks after injection) was 3-5 fold higher for AAV-8 compared with AAV-2. There were no differences in the vector clearance in the semen among rabbits from the low-dose cohorts of AAV-2 and AAV-8. After 12 weeks, all semen samples from all cohorts tested negative. In another rabbit model, vasectomized prior to vector injection, we determined that semen samples lacking germ cells were also positive for vector-DNA sequences. The kinetics of vector clearance in these samples was dose- and time-dependent and serotype-independent. Because the presence of capsid in early-time points is critical for predicting possible immune responses against the viral vector, we determine the vector biodistribution one week after injection of 1×1013 vg/kg of AAV-2 or AAV-8. Rabbits were euthanized and their organs were harvested and analyzed for vector DNA presence through real-time quantitative PCR. Comparing to AAV-2, AAV-8 genomes were 2-5 fold times higher in all organs (spleen, lung, heart, and kidney), with the exception of liver where vector-DNA content was comparable (range from 25-69 copy number/cell). In addition, testes, accessory glands, and prostates were positive for the vector DNA, albeit in very low levels (the highest level of vector DNA found in those organs was 3copy number/cell in the testis of one rabbit injected with AAV-8). These differences may reflect the distributions of cellular receptors for AAV-2 and AAV-8, which may also explain the higher content of vector genome in the semen of high-dose AAV-8 cohort. Together our findings suggest that AAV-8 ensures higher transgene expression than AAV-2 and preexisting immunity to AAV-2, a naturally acquired virus in humans, may not limit AAV-8-mediated gene delivery. The overall kinetics of AAV vector clearance in the semen seems to be independent of the presence of germ cells and vector serotype. However, early biodistribution data of AAV-8 suggests a distinct safety profile from AAV-2.

Characterization of the Relationship of AAV Capsid Domain Swapping to Liver Transduction Efficiency

Recombinant adeno-associated virus (AAV) vectors show promise for use in gene therapy. For liver-targeted gene transfer in animals, AAV vectors pseudotyped with the AAV serotype 8 (AAV8) capsid have definite advantages over the widely used but less efficient serotype AAV2, even though the capsid amino acid sequences are 82% conserved. To demonstrate the mechanism behind the higher liver transduction efficiency associated with AAV8 capsids, we adopted a domain-swapping strategy that would generate 27 chimeric capsid genes containing exchanged domains between AAV2 and AAV8. The resulting chimeric capsids were then used to package AAV genomes with a liver-specific human coagulation factor IX (hFIX) expression cassette. By comparing the transduction efficiencies between vectors pseudotyped with chimeric, AAV2 and AAV8 capsids, we found that the more efficient liver transduction achieved by AAV8 was closely related to the components of its interstrand Loop IV domain, particularly the subloops 1 and 4. These subloops are exposed on opposite sides of a threefold proximal peak on the virion surface, which may function as a critical structural determinant for AAV transduction. Because a single specific peptide component could not explain all the observed differences in the transduction parameters, we suggest that important subloop regions require interaction with other portions of the capsid for their functioning.

Structure of Adeno-Associated Virus Serotype 8, a Gene Therapy Vector

Adeno-associated viruses (AAVs) are being developed as gene therapy vectors, and their efficacy could be improved by a detailed understanding of their viral capsid structures. AAV serotype 8 (AAV8) shows a significantly greater liver transduction efficiency than those of other serotypes, which has resulted in efforts to develop this virus as a gene therapy vector for hemophilia A and familial hypercholesterolemia. Pseudotyping studies show that the differential tissue tropism and transduction efficiencies exhibited by the AAVs result from differences in their capsid viral protein (VP) amino acids. Towards identifying the structural features underpinning these disparities, we report the crystal structure of the AAV8 viral capsid determined to 2.6-A resolution. The overall topology of its common overlapping VP is similar to that previously reported for the crystal structures of AAV2 and AAV4, with an eight-stranded beta-barrel and long loops between the beta-strands. The most significant structural differences between AAV8 and AAV2 (the best-characterized serotype) are located on the capsid surface at protrusions surrounding the two-, three-, and fivefold axes at residues reported to control transduction efficiency and antibody recognition for AAV2. In addition, a comparison of the AAV8 and AAV2 capsid surface amino acids showed a reduced distribution of basic charge for AAV8 at the mapped AAV2 heparin sulfate receptor binding region, consistent with an observed non-heparin-binding phenotype for AAV8. Thus, this AAV8 structure provides an additional platform for mutagenesis efforts to characterize AAV capsid regions responsible for differential cellular tropism, transduction, and antigenicity for these promising gene therapy vectors.

Correction of Clinical Manifestations of Canine Mucopolysaccharidosis I with Neonatal Retroviral Vector Gene Therapy

Mucopolysaccharidosis I (MPS I) (Hurler syndrome) is due to deficient alpha-L-iduronidase (IDUA) activity and is the most common of the MPS disorders. Neonatal MPS I dogs were injected intravenously (IV) with a gamma retroviral vector containing a complete long-terminal repeat (LTR) and an internal human alpha(1)-antitrypsin (hAAT) promoter upstream of the canine IDUA complementary DNA (cDNA). This resulted in stable serum IDUA activity of 366 +/- 344 units (U)/ml (28-fold normal) for up to 1.8 years, which likely derived primarily from secretion of IDUA by transduced liver cells. Retroviral vector (RV)-treated dogs had >18% of normal IDUA activity in organs and had decreased severity and/or incidence of hernias, chest deformities, joint disease, facial dysmorphia, corneal clouding, valvular heart disease, and aortic dilatation as compared with untreated MPS I dogs. The marked reduction that was observed in lysosomal storage in the brain of RV-treated dogs may have been due in part to expression from the LTR of the vector in cells in the brain. This possibility will be explored in future studies, because the potential for insertional mutagenesis has raised concerns about using vectors with an intact LTR. If proven safe, this gene therapy technique may be utilized in treating children with Hurler syndrome.

A Two-hybrid Screen Identifies Cathepsins B and L as Uncoating Factors for Adeno-associated Virus 2 and 8

Vectors based on different serotypes of adeno-associated virus hold great promise for human gene therapy, based on their unique tissue tropisms and distinct immunological profiles. A particularly interesting candidate is AAV8, which can efficiently and rapidly transduce a wide range of tissues in vivo. To further unravel the mechanisms behind AAV8 transduction, we used yeast two-hybrid analyses to screen a mouse liver complementary DNA library for cellular proteins capable of interacting with the viral capsid proteins. In total, we recovered approximately 700 clones, comprising over 300 independent genes. Sequence analyses revealed multiple hits for over 100 genes, including two encoding the endosomal cysteine proteases cathepsins B and L. Notably, these two proteases also physically interacted with the corresponding portion of the AAV2 capsid in yeast, but not with AAV5. We demonstrate that cathepsins B and L are essential for efficient AAV2- and AAV8-mediated transduction of mammalian cells, and document the ability of purified cathepsin B and L proteins to bind and cleave intact AAV2 and AAV8 particles in vitro. These data suggest that cathepsin-mediated cleavage could prime AAV capsids for subsequent nuclear uncoating, and indicate that analysis of additional genes recovered in our screen may help to further elucidate the mechanisms behind transduction by AAV8 and related serotypes.

Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8

It has been recently shown that recombinant adeno-associated virus serotype 8 (rAAV8) is a robust alternative serotype vector that overcomes many of the limitations of rAAV2 and transduces various tissues efficiently and globally through systemic vector administration. AAV9 is a serotype newly isolated from human tissues, but our knowledge of the biology of rAAV9 in vivo is currently limited. Here, we demonstrate by a series of comprehensive side-by-side experiments with rAAV8 and 9 vectors delivered via different routes or at various doses in mice that rAAV9 vectors share the robustness of rAAV8, i.e., (1) very high liver transduction efficiency irrespective of whether vectors are administered intravascularly or extravascularly and (2) substantial transduction in the heart, skeletal muscle, and pancreas by peripheral vein injection. Importantly, rAAV9 transduced myocardium 5- to 10-fold higher than rAAV8, resulting in over 80% cardiomyocyte transduction following tail vein injection of as low as 1.0 × 1011 particles per mouse. Thus rAAV9, as well as rAAV8, is a robust vector for gene therapy applications and rAAV9 is superior to rAAV8 specifically for cardiac gene delivery by systemic vector administration.

746. Localization of Structural Determinants in AAV Capsid for Efficient Liver Transduction by Domain Swapping between AAV-2 and AAV-8

Compared with the AAV-2 serotype, AAV-8 pseudotyped vectors have become another promising candidate for liver-targeted gene delivery due to its ability to transduce almost all hepatocytes in vivo. It has been proposed that the faster uncoating rate of AAV-8 compared to AAV-2 is the key to its more robust transduction efficiencies in liver. However, given the high degree of amino acid conservation, the actual mechanism or relevant structural determinants responsible for these differences are still unsolved.

Production, purification, crystallization and preliminary X-ray analysis of adeno-associated virus serotype 8

Adeno-associated viruses (AAVs) are actively being developed for clinical gene-therapy applications and the efficiencies of the vectors could be significantly improved by a detailed understanding of their viral capsid structures and the structural determinants of their tissue-transduction interactions. AAV8 is approximately 80% identical to the more widely studied AAV2, but its liver-transduction efficiency is significantly greater than that of AAV2 and other serotypes. The production, purification, crystallization and preliminary X-ray crystallographic analysis of AAV8 viral capsids are reported. The crystals diffract X-rays to 3.0 A resolution using synchrotron radiation and belong to the hexagonal space group P6(3)22, with unit-cell parameters a = 257.5, c = 443.5 A. The unit cell contains two viral particles, with ten capsid viral protein monomers per crystallographic asymmetric unit.

504. In Vivo Characterization of AAV Serotype 9 Vectors in Mice

Recombinant AAV(rAAV) vectors are promising vehicles for achieving stable transduction in vivo. We have demonstrated that, in contrast to rAAV2 vectors, which have limited transduction efficiency in many tissues, rAAV8 vectors can transduce all the hepatocytes, cardiomyocytes and skeletal myofibers throughout the body, and a majority of pancreatic acinar cells by peripheral vein injection at a high dose, in mice. AAV serotype 9 has been recently isolated from non-human primate tissues and recombinant vectors based on this serotype have been shown to transduce various tissues with high efficiency. In order to characterize the behavior of rAAV9 vectors in mice, we injected mice with various rAAV9 vectors via several different routes and analyzed transgene expression and tissue distribution. First, we produced a liver specific promoter-driven human factor IX (hFIX)-expressing rAAV9 vector, AAV9-hFIX16, and injected adult C57BL/6 male and female mice via 4 different routes; i.e., portal vein (PV), tail vein (TV), intraperitoneal (IP) and subcutaneous (SC) injections at a dose of 8.0|[times]|10e10 vg/mouse. For comparison, we also injected mice with AAV8-hFIX16 at the same dose. Liver transduction efficiency was monitored by measuring hFIX levels in the plasma. Two weeks post-injection, PV injection with the rAAV9 vector (9PV), 9TV and 9IP achieved comparable hFIX levels of 350-400 |[mu]|g/ml, which was slightly higher than the mice injected with the rAAV8 vector via PV (8PV). SC injection achieved approximately half of the levels with both AAV9 and AAV8 vectors, but still expressed over 100 |[mu]|g/ml of hFIX in plasma at this dose. Liver transduction in females was lower than males by |[sim]|30% with both serotypes as seen with rAAV2 vectors in this mouse strain. Interestingly, TV and IP injections of the rAAV9 vector showed slower kinetics of hFIX expression compared to the PV injection, even though any of these routes achieved a comparable level of expression 2 weeks-post injection. Such distinct expression kinetics depending on the route of vector administration was not observed with rAAV8 vector. In the second experiment, we injected C57BL/6 mice with rAAV9 vectors expressing a lacZ marker gene; i.e., AAV9-EF1|[alpha]|-nlslacZ or AAV9-CMV-lacZ via TV at two different doses, 3.0|[times]|10e11 and 1.8|[times]|10e12 vg/mouse (n=2 per group, total 8 mice for 4 groups). Development of anti-|[acirc]| galactosidase antibody was monitored by ELISA. The antibody was not detected in any of the 8 mice 7 days post-injection, but detected in 3 of 4 mice injected with AAV9-CMV-lacZ 10 days post-injection, when all the 8 mice were sacrificed for tissue distribution analysis. Liver transduction efficiency with the CMV vector was 3% at 3.0|[times]|10e11 and reached 90% at 1.8|[times]|10e12 vg/mouse. The tissue distribution pattern with rAAV9 vectors was similar to that with rAAV8 vectors, in that, in addition to the liver, they transduced heart, skeletal muscle and pancreatic acinar cells with high efficiency. Of note is that rAAV9 had greater transduction efficiency in the heart than rAAV8, and 3.0|[times]|10e11 vg/mouse was enough to transduce all the cardiomyocytes via TV injection of rAAV9. Thus, rAAV9, as well as rAAV8, is a robust vector for gene therapy applications.

38. Inhibition of Apolipoprotein B-100 Expression by Lentivirus Mediated RNA Interference in Mice after Stem Cell Transduction and Transplantation

Apolipoprotein B-100 (ApoB-100) is an essential component low density lipoprotein (LDL), transporting cholesterol in circulation and acting as a ligand for the LDL-receptor. In atherosclerotic mice the reduction of ApoB-100 expression leads to the reduction of plasma LDL-cholesterol levels and delays the development of atherosclerotic plaques in the vessel wall. By using highly efficient RNA interference method it could be possible to treat lipid disorders by gene therapy if an efficient liver transduction is achieved. Contribution of different stem cell populations to regenerating mouse livers is not fully understood, but it has been shown that stem cells do take part in the regeneration of the liver and that functional therapeutic genes can be efficiently incorporated by ex vivo gene transfer followed by transplantation.

87. Determinants of Toxicity Induced by In Vivo Expression of Short Hairpin RNA from AAV Vectors

Double-stranded (ds) vectors based on Adeno-associated virus (AAV) are promising candidates for the in vivo delivery of short hairpin RNA (shRNA), as they give robust and stable gene expression, and lack any inherent toxicity. In efforts to engineer this vector towards clinical use, we evaluated a battery of dsAAVs, expressing shRNAs directed against transcripts from two different targets, Hepatitis B virus (HBV) or the human alpha-1-antitrypsin (hAAT) gene. We generated four individual vectors against each target, expressing shRNAs with stems ranging from 19 to 25 nucleotides in length. Each vector was pseudotyped with AAV-8 capsids for efficient liver transduction, and then injected into mice transgenic for the HBV or hAAT gene(s), respectively.

531. Balloon Catheter-Mediated Hepatic Vein Delivery of a Viral Vector Mitigates Neutralization by Anti-Viral Antibodies and Results in Efficient Transduction of Rabbit Liver

The liver, and in particular, hepatocytes, represent an attractive target for both viral and non-viral gene transfer vectors. However, there are certain delivery issues associated with both of these vector systems that make their use in humans problematic. In particular, for viral vectors, the existence of anti-viral antibodies in the general population represents a potential barrier to their effective use in the clinic. Building upon our earlier success using balloon catheters to deliver plasmid DNA by way of the hepatic venous circulation to rabbit liver, we have used this same model to ask if we could use a similar approach to deliver a model viral vector. Specifically, we have compared a local, balloon catheter-mediated hepatic vein delivery protocol to systemic delivery of a CMV-driven adenoviral vector expressing b-galactosidase in terms of liver transduction efficiency, toxicity, and cell types transduced. We have also made this comparison in the presence of defined anti-AdV antibody titers in the recipient rabbits. In the naive animal, local balloon catheter-based delivery conferred an advantage in overall liver transduction when compared to systemic delivery of an identical dose of virus. In rabbits bearing anti-AdV antibody titers equivalent to those found in pooled human serum, this difference in expression between local and systemic delivery was even more striking. Importantly, in the presence of passively-administered anti-AdV antibodies, balloon-catheter mediated delivery resulted in expression levels that were comparable to those obtained by systemic delivery of an equivalent dose in a naive animal. Since in general, systemic delivery of AdV in naive animal models results in expression levels of secreted proteins regarded as therapeutic, the present results predict that retrograde delivery by a hepatic vein route using a balloon catheter should result in therapeutic levels of expression, even in the presence of average human levels of anti-AdV antibodies. Further support for this hypothesis is the finding that this local hepatic vein delivery approach resulted in the majority of expression originating from hepatocytes even in the passively immunized animals. In contrast, systemic delivery of AdV in passively immunized animals resulted in the majority of expression originating from non-hepatocytes. This latter finding implies that this local approach should help minimize the transduction of antigen-presenting cells, thereby reducing any immune consequences of viral transduction. Taken together, these data suggest that coupled with additional features such as a hepatocyte-specific expression cassette, balloon catheter viral-mediated transduction of the liver may represent a practical clinical approach for treating indications for which hepatocytes can be used as a depot for therapeutic protein production.

905. Hydrodynamic Injection of Helper-Dependent Adenoviral Vectors Increases Liver Transduction Efficiency and Decreases Acute Inflammatory Response

Hydrodynamic injection of helper-dependent adenoviral vectors (HDAd) in mice results in increased hepatic transduction resulting in higher levels of long-term transgene expression compared to conventional injection. Furthermore, hydrodynamic injection resulted in reduced splenic and pulmonary transduction and reduced serum levels of the proinflammatory cytokines IL-6 and IL-12. Interestingly, hydrodynamic injection does not appear to alter Kupffer cell uptake of the vector compared to conventional injection. Taken together, these results strongly suggest that hepatic transduction by HDAd, at least alone, does not necessarily provoke a severe innate inflammatory response, but that systemic vector dissemination may play a major role in the severity of the innate inflammatory response. These results further suggest that the safety and efficacy of HDAd-mediated, liver-directed gene therapy may be improved if the vector could be preferentially, if not exclusively, targeted to hepatocytes.

Input Virion Proteins: Cryptic Targets of Antivector Immune Responses in Preimmunized Subjects

Input Virion Proteins: Cryptic Targets of Antivector Immune Responses in Preimmunized Subjects

789. Hydrodynamic Injection of Helper Dependent Adenoviral Vectors Increases Liver Transduction Efficiency and Decreases Acute Inflammatory Response

Top of pageAbstract Hydrodynamic injection of helper–dependent adenoviral vectors (HDAd) in mice results in significantly higher efficiency hepatic transduction and significantly higher levels of transgene expression compared to conventional injection without compromising duration of expression. Furthermore, significantly less splenic transduction was observed following hydrodynamic injection compared to conventional injection. These results suggest that hydrodynamic injection alters vector biodistribution to favor transduction of the liver and consequently reduce transduction of other organs. Moreover, elevations in IL–6 and IL–12 were less following hydrodynamic than conventional injection suggesting that activation of the innate inflammatory response may be reduced by minimizing systemic exposure/transduction through preferential hepatic transduction. Although systemic hydrodynamic injection is not possible in large animals, localized hydrodynamic injection may provide a method of achieving efficient HDAd–mediated hepatic transduction and minimizing the acute inflammatory immune response.