Vectors For Liver-directed Gene Therapy Research Articles

Restoring the natural tropism of AAV2 vectors for human liver.

Recent clinical successes in gene therapy applications have intensified interest in using adeno-associated viruses (AAVs) as vectors for therapeutic gene delivery. Although prototypical AAV2 shows robust in vitro transduction of human hepatocyte-derived cell lines, it has not translated into an effective vector for liver-directed gene therapy in vivo. This is consistent with observations made in Fah-/-/Rag2-/-/Il2rg-/- (FRG) mice with humanized livers, showing that AAV2 functions poorly in this xenograft model. Here, we derived naturally hepatotropic AAV capsid sequences from primary human liver samples. We demonstrated that capsid mutations, likely acquired as an unintentional consequence of tissue culture propagation, attenuated the intrinsic human hepatic tropism of natural AAV2 and related human liver AAV isolates. These mutations resulted in amino acid changes that increased binding to heparan sulfate proteoglycan (HSPG), which has been regarded as the primary cellular receptor mediating AAV2 infection of human hepatocytes. Propagation of natural AAV variants in vitro showed tissue culture adaptation with resulting loss of tropism for human hepatocytes. In vivo readaptation of the prototypical AAV2 in FRG mice with a humanized liver resulted in restoration of the intrinsic hepatic tropism of AAV2 through decreased binding to HSPG. Our results challenge the notion that high affinity for HSPG is essential for AAV2 entry into human hepatocytes and suggest that natural AAV capsids of human liver origin are likely to be more effective for liver-targeted gene therapy applications than culture-adapted AAV2.

Autophagy determines efficiency of liver-directed gene therapy with adeno-associated viral vectors.

Use of adeno‐associated viral (AAV) vectors for liver‐directed gene therapy has shown considerable success, particularly in patients with severe hemophilia B. However, the high vector doses required to reach therapeutic levels of transgene expression caused liver inflammation in some patients that selectively destroyed transduced hepatocytes. We hypothesized that such detrimental immune responses can be avoided by enhancing the efficacy of AAV vectors in hepatocytes. Because autophagy is a key liver response to environmental stresses, we characterized the impact of hepatic autophagy on AAV infection. We found that AAV induced mammalian target of rapamycin (mTOR)–dependent autophagy in human hepatocytes. This cell response was critically required for efficient transduction because under conditions of impaired autophagy (pharmacological inhibition, small interfering RNA knockdown of autophagic proteins, or suppression by food intake), recombinant AAV‐mediated transgene expression was markedly reduced, both in vitro and in vivo. Taking advantage of this dependence, we employed pharmacological inducers of autophagy to increase the level of autophagy. This resulted in greatly improved transduction efficiency of AAV vectors in human and mouse hepatocytes independent of the transgene, driving promoter, or AAV serotype and was subsequently confirmed in vivo. Specifically, short‐term treatment with a single dose of torin 1 significantly increased vector‐mediated hepatic expression of erythropoietin in C57BL/6 mice. Similarly, coadministration of rapamycin with AAV vectors resulted in markedly enhanced expression of human acid‐α‐glucosidase in nonhuman primates. Conclusion: We identified autophagy as a pivotal cell response determining the efficiency of AAVs intracellular processing in hepatocytes and thus the outcome of liver‐directed gene therapy using AAV vectors and showed in a proof‐of‐principle study how this virus–host interaction can be employed to enhance efficacy of this vector system. (Hepatology 2017;66:252–265).

Helper-dependent adenoviral vectors for liver-directed gene therapy of primary hyperoxaluria type 1.

Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to deficiency of the peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT) which catalyzes conversion of glyoxylate into glycine. AGT deficiency results in overproduction of oxalate which ultimately leads to end-stage renal disease and death. Organ transplantation as either preemptive liver transplantation or combined liver/kidney transplantation is the only available therapy to prevent disease progression. Gene therapy is an attractive option to provide an alternative treatment for PH1. Towards this goal, we investigated helper-dependent adenoviral (HDAd) vectors for liver-directed gene therapy of PH1. Compared to saline controls, AGT-deficient mice injected with an HDAd encoding the AGT under the control of a liver-specific promoter showed a significant reduction of hyperoxaluria and less increase of urinary oxalate following challenge with Ethylene Glycol (EG), a precursor of glyoxylate. These studies may thus pave the way to clinical application of HDAd for PH1 gene therapy.

703. Liver-Directed Gene Therapy for Primary Hyperoxaluria Type 1

Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to deficiency of peroxisomal enzyme alanine:glyoxylate-aminotransferase (AGT) which catalyzes the conversion of glyoxylate to glycine. In PH1 patients, glyoxylate cannot be converted into glycine and is oxidized to oxalate resulting in hyperoxaluria. The excess of oxalate causes deposition of insoluble calcium oxalate in the kidney and other tissues leading to nephrolithiasis, nephrocalcinosis, kidney failure, and systemic tissue damage. Combined liver/kidney transplantation is the only available therapeutic strategy for disease treatment. Gene therapy is an attractive option to provide a definitive cure for PH1. Towards this goal, we investigated helper-dependent adenoviral (HDAd) vectors for liver-directed gene therapy of PH1. We injected PH1 mice with an HDAd encoding AGT under the control of a liver-specific promoter and observed normalization of urinary oxalate at the doses of 5×10e12 and 1×10e13 vector particles (vp)/kg and partial correction with 1×10e12 vp/kg. Following challenge with Ethylene Glycol (EG), a precursor of glyoxylate, we observed reduced elevations of urinary oxalate in HDAd-injected mice compared to saline controls.Next, we hypothesized that overexpression of glyoxylate reductase/hydroxypyruvate reductase (GRHPR) or glutamate-pyruvate transaminase (GPT) results in reduction of hyperoxaluria in PH1 by steering glyoxylate towards glycolate synthesis or transamination, respectively. To test this hypothesis, we injected PH1 mice with HDAd vectors expressing GRHPR or GPT. Both vectors resulted in significant reduction of hyperoxaluria and co-injection of the two vectors resulted in long-term normalization of oxalate excretion. Therefore, metabolic diversion towards non-toxic metabolites has potential for treatment of hyperoxaluria and vector-mediated GRHPR and/or GPT overexpression may be an alternative or adjunctive strategy to enhance efficiency of gene replacement therapy for PH1.We have recently developed a minimally invasive method to improve the therapeutic index of HDAd based on balloon occlusion catheter to achieve preferential delivery of the vector to the liver (Brunetti-Pierri et al., 2009 and 2012). This method may permit in humans correction of PH1 using clinically relevant lower doses of HDAd. Based on risk:benefit assessment, PH1 is an attractive disease for clinical application of this method.

AAV8 capsid variable regions at the two-fold symmetry axis contribute to high liver transduction by mediating nuclear entry and capsid uncoating

Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8׳s robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 – a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII & IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole liver cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype.

Helper-dependent adenoviral vectors for liver-directed gene therapy

Helper-dependent adenoviral (HDAd) vectors devoid of all viral-coding sequences are promising non-integrating vectors for liver-directed gene therapy because they have a large cloning capacity, can efficiently transduce a wide variety of cell types from various species independent of the cell cycle and can result in long-term transgene expression without chronic toxicity. The main obstacle preventing clinical applications of HDAd for liver-directed gene therapy is the host innate inflammatory response against the vector capsid proteins that occurs shortly after intravascular vector administration resulting in acute toxicity, the severity of which is dependent on vector dose. Intense efforts have been focused on elucidating the factors involved in this acute response and various strategies have been investigated to improve the therapeutic index of HDAd vectors. These strategies have yielded encouraging results with the potential for clinical translation.

Improved Hepatic Transduction, Reduced Systemic Vector Dissemination, and Long-Term Transgene Expression by Delivering Helper-Dependent Adenoviral Vectors into the Surgically Isolated Liver of Nonhuman Primates

Helper-dependent adenoviral vectors (HDAds) are attractive vectors for liver-directed gene therapy because they can mediate sustained, high-level transgene expression without chronic toxicity. However, high vector doses are required to achieve efficient hepatic transduction by systemic delivery because of a nonlinear dose response. Unfortunately, such high doses result in systemic vector dissemination and dose-dependent acute toxicity with potentially severe and lethal consequences. We hypothesize that the threshold to efficient hepatic transduction may be circumvented by delivering the vector into the surgically isolated liver via the portal vein. Total hepatic isolation was achieved by occluding hepatic inflow from the portal vein and hepatic artery and by occluding hepatic venous outflow at the inferior vena cava. We demonstrate in nonhuman primates that this approach resulted in significantly higher efficiency hepatic transduction with reduced systemic vector dissemination compared with systemic intravascular delivery. This method of delivery was associated with transient acute toxicity, the severity of which was variable. Importantly, stable, high levels of transgene expression were obtained for at least 665 days for one baboon and for at least 560 days for two baboons with no evidence of long-term toxicity.

904. High Efficiency Hepatic Transduction and Long-Term Transgene Expression by Delivering Helper-Dependent Adenoviral Vectors into the Surgically Isolated Liver of Nonhuman Primates

Helper-dependent adenoviral vectors (HDAd) are attractive vectors for liver-directed gene therapy because they can mediate long-term, high level transgene expression with no long-term toxicity. However, as a consequence of a threshold effect, high vector doses are required to achieve efficient hepatic transduction by peripheral intravenous injection which unfortunately results in a dose-dependent activation of the innate inflammatory response. Clearly strategies to overcome the threshold to efficient hepatic transduction are needed to improve the therapeutic index of HDAd. We hypothesized that this obstacle could be surmounted by delivering the vector exclusively to the liver. To test this hypothesis, we have injected HDAd directly into the surgically isolated liver via the portal vein in nonhuman primates. Total hepatic isolation was achieved by occluding hepatic inflow from the portal vein and hepatic artery and by occluding hepatic venous outflow at the inferior vena cava. Prior to total hepatic isolation, saline was infused into the portal vein to flush blood out of the liver. The vector was then injected directly into the liver via the portal vein and allowed to dwell for 30 minutes, following which unabsorbed vector was flushed out via a catheter placed in the vena cava to minimize systemic vector dissemination. Our results revealed that significantly higher hepatic transduction efficiencies can be achieved with relatively low vector doses compared to peripheral intravenous injection. Importantly, stable, high levels of transgene expression were observed for up to one year with no long-term toxicity. This approach may increase the safety and efficacy of HDAd-mediated, liver-directed gene therapy by minimizing the dose required to achieve efficient hepatic transduction and by minimizing systemic vector dissemination.

12. Self-Complementary AAV2 Vectors Transduce Liver with the Same Efficiency as AAV8: The Critical Role of Second-Strand Synthesis in AAV Biology

Top of pageAbstract Previous studies have shown that conventional single-stranded (ss) AAV-2 vectors only transduce a limited number of liver cells, which restricts the use of AAV-mediated liver gene transfer. Recently, we demonstrated that self-complementary (sc) AAV vector significantly increased liver transduction by overcoming the rate-limiting step of second-strand synthesis. Additional studies have shown that cross-packaging of rAAV-2 genome into specific serotype AAV capsids (e.g. AAV6 and AAV8) also increase liver transduction. Although the exact mechanism is not totally clear, the increased transduction by specific serotype AAVs over AAV2 might be related to differences in virus binding, intracellular trafficking, nuclear entry, uncoating or all of the above. In this study, we demonstrate that scAAV genomes packaged into serotypes other than type 2 are also superior to their single-strand counterparts in liver transduction. In addition, we extend these studies to determine if serotype scAAVs show the same hierarchy in transgene expression as that of ssAAV counterparts. 1|[times]|1011 ss vs sc vector genomes carrying liver-specific expression cassette of human factor IX (hFIX) were packaged into serotype 2, 6 and 8 and injected via portal vein into the livers of C57BL/6 mice. Expression of hFIX was monitored over a time course lasting eight weeks. The results showed that among ss vectors, AAV8 gave the strongest expression, followed by AAV6, while AAV2 gave much lower expression at all time points. For each serotype, higher expression of hFIX protein was achieved by sc vector over its ss counterpart. However, the increase of expression by scAAV6 or scAAV8 was not as significant as that of scAAV2 (3 fold vs 50 fold respectively). Interestingly, both ss and scAAV2 exhibited a unique kinetics of hFIX expression, which was distinctly different from AAV6 and 8. hFIX expression by AAV2 increased slowly during eight weeks post injection, while AAV6 and AAV8 reached their peaks rapidly. As a result, at two weeks post injection, scAAV8 and scAAV6 expressed more efficiently than scAAV2, however, scAAV2 exceeded scAAV6 after 4 weeks and was equal to scAAV8 by 8 weeks. Based on these results, the total number of AAV2 virus that enter hepatocyte nuclei is not less than that of AAV8 or AAV6 even though there is a lag phase for expression from AAV2 virions. The unique expression profile of AAV2 might reflect a step post viral binding to cell surface that is inefficient. Our results clearly demonstrate that scAAV-2 will perform equivalent to type 8 in mouse liver. Unlike AAV serotype 8, which has been reported to have the property of wide spread dissemination to other organs, the well-characterized AAV2 vector carrying sc genomes may still be among the most promising candidate vectors for liver-directed gene therapy.

906. High Efficiency Hepatic Transduction with Minimal Toxicity Following “Hydrodynamic” Injection of HDAd into Nonhuman Primates

Helper-dependent adenoviral vectors (HDAd) are attractive vectors for liver-directed gene therapy because they can mediate sustained, high level transgene expression with no long-term toxicity. However, as a consequence of a threshold effect, high vector doses are required to achieve efficient hepatic transduction by peripheral intravenous injection which unfortunately results in a dose-dependent activation of the innate inflammatory response. Clearly strategies to overcome the threshold to efficient hepatic transduction are needed to improve the therapeutic index of HDAd. We have recently shown that systemic hydrodynamic injection of HDAd into mice resulted in exceedingly high levels of hepatic transduction and reduction in both systemic vector dissemination and acute proinflammatory cytokines compared to conventional injection. Hydrodynamic injection offers a method of dramatically increasing the therapeutic index of HDAd but, unfortunately, cannot be applied to larger animals due to the large injection volume. Therefore, we have developed a minimally invasive method to mimic hydrodynamic injection in nonhuman primates which does not require injection of large volumes. This method involves the use of balloon occlusion catheters percutaneously positioned in the vena cava to transiently obstruct hepatic venous outflow to increase the intrahepatic pressure prior to simple peripheral intravenous injection of HDAd in a small volume. We demonstrate in nonhuman primates that this novel method of vector delivery results in an exceedingly high and unprecedented level of hepatic transduction with low vector doses resulting in stable, long-term transgene expression. This was accompanied by reduced systemic vector dissemination and minimal, transient elevations of proinflammatory cytokines. Despite the relatively high intrahepatic pressures achieved, there was minimal evidence of liver injury; no histological abnormalities were observed and only minimal and transient elevation of AST and ALT were noted. The procedure itself was uneventful and very well tolerated with no clinical manifestations of acute or chronic toxicities. This novel method of delivering HDAd is simple, minimally invasive, and clinically relevant since it increases the therapeutic index of HDAd for liver-directed gene therapy by allowing high efficiency hepatic transduction with low vector doses and with negligible acute or chronic toxicities.

173. Adenovirus-Mediated Gene Transfer to Mouse and Dog Pancreas In Vivo

Helper-dependent adenoviral vectors (HDAd) are attractive vectors for liver-directed gene therapy because they can mediate sustained, high level transgene expression with negligible long-term toxicity. However, high vector doses are required to achieve efficient hepatic transduction by peripheral intravenous injection due to a nonlinear dose response. Unfortunately, such high systemic doses result in wide spread vector dissemination and dose-dependent activation of the acute inflammatory response which can result in severe and lethal acute toxicity. We hypothesize that this problem can be surmounted by delivering the vector exclusively to the liver thereby permitting high efficiency hepatic transduction with low vector doses and with minimal systemic vector dissemination. To test this hypothesis, we injected HDAd directly into the surgically isolated liver via the portal vein in nonhuman primates. Total hepatic isolation was achieved by occluding hepatic blood inflow from the portal vein and hepatic artery and by occluding hepatic blood outflow at the vena cava. Prior to total hepatic isolation, saline was infused into the portal vein to flush blood out of the liver. The vector was then injected directly into the isolated liver via the portal vein and allowed to dwell for 30 min to prolong exposure to hepatocytes. Following the 30 min dwell time, unabsorbed vector was removed from the liver to minimize systemic dissemination. This was accomplished by infusing saline into the portal vein and collecting the unabsorbed vector from a vena cava catheter. We investigated the safety, feasibility and efficacy of this approach and our results revealed that the surgical procedure was well tolerated in nonhuman primates and that significantly higher hepatic transduction efficiencies can be achieved with relatively low vector doses as compared to those obtained by peripheral intravenous injection. This approach may increase the safety and efficacy of HDAd-mediated, liver-directed gene therapy by minimizing the dose required to achieve efficient hepatic transduction and by minimizing systemic vector dissemination.

Acute toxicity after high-dose systemic injection of helper-dependent adenoviral vectors into nonhuman primates.

Systemic intravascular delivery of adenoviral (Ad) vectors for liver-directed gene therapy has been widely employed because of its simplicity, noninvasiveness, and potential for high transduction. For first-generation Ad vectors (FGAd), this results in high but transient levels of transgene expression and long-term hepatotoxicity due to viral gene expression from the vector backbone. Furthermore, high doses also result in an acute innate inflammatory response with potentially lethal consequences. Unlike FGAd, helper-dependent Ad vectors (HDAd) contain no viral genes and can provide sustained, high-level transgene expression with negligible long-term toxicity. However, whether the absence of viral gene expression leads to any decrease of acute toxicity in nonhuman primates has yet to be determined. To address this, we injected one baboon with 5.6 x 10(12) HDAd viral particles (VP)/kg and a second with 1.1 x 10(13) VP/kg. Approximately 50% hepatocyte transduction, accompanied by mild and transient acute toxicity, was observed in the animal receiving the lower dose. In the animal receiving the higher dose, 100% hepatocyte transduction, accompanied by lethal acute toxicity, was observed. These results indicate that systemic delivery of HDAd, like FGAd, results in acute toxicity in baboons consistent with activation of the innate inflammatory response, the severity of which is dose dependent, and confirm the hypothesis that Ad-mediated acute toxicity is independent of viral gene expression.

Presence of replicating virus in recombinant hepadnavirus stocks results from recombination and can be eliminated by the use of a packaging cell line.

Mutant hepatitis B viruses are useful tools to study the viral life cycle and viral pathogenesis. Furthermore, recombinant hepatitis B viruses are candidate vectors for liver-directed gene therapy. Because wild-type viruses present in recombinant or mutant virus stocks may falsify experimental results and are detrimental for a viral vector, we investigated whether and to what extent wild-type virus is present in recombinant virus stocks and where it originates from. We took advantage of the duck model of hepatitis B virus infection which allows very sensitive detection of replication-competent viruses by infection of primary duck hepatocytes or of ducklings in vivo. Recombinant hepatitis B virus stocks contained significant amounts of wild-type viruses, which were most probably generated by homologous recombination between plasmids containing homologous viral sequences. In addition, replication-competent viral genomes were reconstituted from plasmids which contained replication-deficient but redundant viral sequences. Using a stable cell line for packaging of deficient viral genomes, no wild-type virus was detected, neither by infection of primary hepatocytes nor in vivo.

Usefulness of Intra-arterial Embolization Method Using Gelfoam Particles in Effective Gene Transduction of Adenoviral Vector for Liver-directed Gene Therapy: an Preliminary Animal Study in Dogs

Usefulness of Intra-arterial Embolization Method Using Gelfoam Particles in Effective Gene Transduction of Adenoviral Vector for Liver-directed Gene Therapy: an Preliminary Animal Study in Dogs

A replication-deficient rSV40 mediates liver-directed gene transfer and a long-term amelioration of jaundice in gunn rats.

In the quest for a recombinant viral vector for liver-directed gene therapy that would permit both prolonged and efficient transgene expression in quiescent hepatocytes in vivo and repeated administration, we evaluated a recombinant simian virus 40 (rSV40). The rSV40 was generated through replacement of the DNA encoding for the T antigens (Tag) by the coding region of human bilirubin-uridine 5'-diphosphate-glucuronosyl-transferase (BUGT) complementary DNA (SV-hBUGT). Helper-free rSV40 units were generated at infectious titers of 5 x 10(9) to 1 x 10(10) infectious units (IU)/mL in a Tag-producing packaging cell line (COS-7 cells). After 1, 3, or 7 daily infusions of 3 x 10(9) IU of SV-hBUGT through an indwelling portal vein catheter in bilirubin-UGT-deficient jaundiced Gunn rats, mean serum bilirubin concentrations decreased by 40%, 60% and 70%, respectively, in 3 weeks and remained at those levels throughout the duration of the study (40 days). Results of liver biopsies from SV-hBUGT-treated Gunn rats, but not from controls, were positive for human BUGT DNA, messenger RNA, and protein. Bilirubin-UGT activity in liver homogenates was 8%-12% of normal, and bilirubin glucuronides were excreted in bile. Immunostaining showed that >50%-60% of hepatocytes stably expressed the transgene. Portal vein infusion of an rSV40 expressing hepatitis B surface antigen (HBsAg) in a naive Gunn rat and a Gunn rat that had received 7 injections of SV-BUGT resulted in approximately equal levels of hepatic expression of HBsAg, indicating that multiple inoculations of SV-BUGT did not elicit neutralizing antibodies. Plasma alanine aminotransferase levels and liver histology remained normal despite repeated injections of rSV40. rSV40 vectors may represent a significant advance toward gene therapy for metabolic diseases.

Retroviral vectors for liver-directed gene therapy.

Retroviruses are popular gene therapy vectors because they stably integrate the DNA copy of their genome into the host chromosome during their replication cycle. The widely used murine retroviral vector systems have two components: the transfer vector for the transgene carries all the cis-acting elements necessary for the replication and efficient integration of the viral DNA; and the packaging cell line produces all the trans-acting proteins necessary for both structural and catalytic functions of the virus. Advances in design of retroviral vectors have resulted in greater degree of biosafety, expanded host range, and increased stability of the virus particles. Retroviral vectors have been widely used in the ex vivo gene therapy protocols to correct the liver diseases in a wide variety of species. In a limited number of applications, in vivo gene therapy has been achieved after the liver cells have been stimulated to regenerate. One major limitation of murine retroviral vectors is their inability to infect nondividing cells. This problem has been overcome by deriving vectors from lentiviruses (a class of retroviruses) that have the ability to infect both dividing and nondividing cells. The lentiviral vectors are derived from human immunodeficiency virus type 1 (HIV-1). Initial studies using lentiviral vectors for gene delivery to the liver in vivo show promising results. A highly crippled version of lentivirus has been generated by using producer cells in which the trans-acting components are expressed by several different coding elements and vectors that incorporate features of self-inactivation. These improvements should ensure biosafety of lentiviruses and make them useful in efficient delivery of therapeutic genes to nondividing differentiated tissues such as the liver.

Adeno-associated virus as a vector for liver-directed gene therapy.

Factors relevant to the successful application of adeno-associated virus (AAV) vectors for liver-directed gene therapy were evaluated. Vectors with different promoters driving expression of human alpha-1-antitrypsin (alpha-1AT) were injected into the portal circulation of immunodeficient mice. alpha-1AT expression was stable but dependent on the promoter. Southern analysis of liver DNA revealed approximately 0.1 to 2.0 provirus copies/diploid genome in presumed head-to-tail concatamers. In situ hybridization and immunohistochemical analysis revealed expression in approximately 5% of hepatocytes clustered in the pericentral region. These results support the use of AAV as a vector for diseases treatable by targeting of hepatocytes.

Gene transfer into hepatocytes and human liver tissue by baculovirus vectors.

Gene therapy of liver diseases requires the development of efficient vectors for gene transfer in vivo. Retroviral and adenoviral vectors have been shown to deliver genes efficiently into hepatocytes in vitro and in vivo. However, these vectors do not allow for exclusive infection of the liver which would be highly advantageous for in vivo gene therapy strategies. We have recently demonstrated that genetically modified baculoviruses (Autographa californica nuclear polyhedrosis virus) efficiently deliver genes into cultured cells and have a strong preference for hepatocytes of different origin. Baculoviral gene transduction efficiency into human hepatocytes was determined to approach 100% and expression levels are high, provided that gene expression is controlled by mammalian promoters. In this report, we present further properties of baculoviruses regarding their use for hepatocyte gene transfer. Baculovirus-mediated gene expression declines rapidly in the hepatocellular carcinoma cell line Huh7 and more slowly in primary cultures of mouse hepatocytes. Direct application of baculoviruses for gene delivery to the liver in vivo is hampered by serum components, presumably by complement. However, we demonstrate here that baculoviral gene transfer is feasible in ex vivo perfused human liver tissue. This result suggests the development of a strategy using baculoviral vectors for liver-directed gene therapy.