Liver Transduction Research Articles

96. Transgene Expression from Recombinant AAV-8 Vectors in the Liver Occurs Independently of the Serotype Origin of the Viral Inverted Terminal Repeats

We present data from the long-term in vivo evaluation of gene transfer vectors combining properties from seven different serotypes of Adeno-Associated Virus (AAV), and discuss how they corroborate our current model for liver transduction by AAV.

319. Improvement of Transgene Expression through the Direct Intrahepatic Injection of Adenoviral Vectors

Top of pageAbstract Numerous diseases could be potentially treated using gene therapy based strategies. An ideal gene therapy system should lead to a safe, low toxicity, long termed and high-level of expression of the therapeutic protein. However, the scaling up of the production for its application to general recombinant viral vector clinical treatment in humans presents a big problem for some of them, for example the third generation adenovirus. Focusing on the improvement of the high-level transgene expression of an adenoviral vector, we characterized a simple and efficient technique to transduce the liver.First and third generation adenovirus were used in mice to characterize the administration of the virus by direct intrahepatic injection. First and third generation adenovirus expressing human interleukin twelve (hIL12), as reporter gene, were assayed in mice by intravenous (IV) and intrahepatic (IH) administration. The expression of the transgene in the first generation adenovirus is under control of the CMV promoter. The third generation adenovirus, contains the hIL-12 gene under the control of an antiprogesterone receptor promoter system. In order to study the relevance of Kupffer cells in the adenovirus liver transduction, we injected both vectors after a clodronate containing liposomes injection. The IH injection of 1×1010 pfu of the first generation adenovirus Ad-IL-12 shows protein levels 2–3 times higher than the administration of the same dose by conventional IV administration. Il-12 expression it is not detected in mice receiving 109 pfu of the Ad-hIL-12 I.V., however the same dose administerred I.H. resulted in clearly detectable levels of the transgene in serum. That should result in an at least 2–3 times lower dose of virus needed to get an identical expression level. The previous injection of clodronate-liposomes improves substantially the expression in both routes and maintains the previously described effect. Moreover the kinetic of the transgene expression is different depending on the route of administration, after IH. administration the highest expression level of the transgene is detected at early time points and an abrupt progressive decrease, on the other hand adenovirus IV administerred showed the peak of transgene expression 48 hours after viral injection. The third generation adenovirus injected intrahepatically offers a better increase in protein expression, 3–4 times, respect to the IV injection. However the empty-liposome preinjection decreases this improvement and the clodronate-liposomes administration inverts this tendency, being the IV better than the IH, in this case. This method evades the phagocytosis by the liver resident macrophages and permits a good expression at low virus doses. Empty-liposomes poorly and the clodronate-liposomes clearly deplete the Kupffer cells and eliminate this difference. This novel technique of gene therapy administration offers several advantages. It makes feasible the administration of less adenovirus that expresses high levels of transgene. It presents a quick and easy treatment method for humans and superior mammals, especially for third generation adenovirus.

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.

Long-term correction of hyperphenylalaninemia by AAV-mediated gene transfer leads to behavioral recovery in phenylketonuria mice.

Classical phenylketonuria (PKU) is a metabolic disorder caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). If untreated, accumulation of phenylalanine will damage the developing brain of affected individuals, leading to severe mental retardation. Here, we show that a liver-directed PAH gene transfer brought about long-term correction of hyperphenylalaninemia and behavioral improvement in a mouse model of PKU. A recombinant adeno-associated virus (AAV) vector carrying the murine PAH cDNA was constructed and administered to PAH-deficient mice (strain PAH(enu2)) via the portal vein. Within 2 weeks of treatment, the hyperphenylalaninemic phenotype improved and completely normalized in the animals treated with higher vector doses. The therapeutic effect persisted for 40 weeks in male mice, while serum phenylalanine concentrations in female animals gradually returned to pretreatment levels. Notably, this long-term correction of hyperphenylalaninemia was associated with a reversal of hypoactivity observed in PAH(enu2) mice. While locomotory activity over 24 h and exploratory behavior were significantly decreased in untreated PAH(enu2) mice compared with the age-matched controls, these indices were completely normalized in 12-month-old male PKU mice with lowered serum phenylalanine. These results demonstrate that AAV-mediated liver transduction ameliorated the PKU phenotype, including central nervous system dysfunctions.

Site and mechanism of leptin action in a rodent form of congenital lipodystrophy.

Lipodystrophy is characterized by the complete or partial absence of adipose tissue, insulin resistance, hepatic steatosis, and leptin deficiency. Here, we show that low-dose central leptin corrects the insulin resistance and fatty liver of lipodystrophic aP2-nSREBP-1c mice, while the same dose given peripherally does not. Central leptin also repressed stearoyl-CoA desaturase-1 (SCD-1) RNA and enzymatic activity, which were increased in livers of lipodystrophic mice. aP2-nSREBP-1c mice homozygous for an SCD-1 deletion had markedly reduced hepatic steatosis, increased saturated fatty acids, decreased acetyl-CoA carboxylase activity, and decreased malonyl-CoA levels in the liver. Despite the reduction in hepatic steatosis, these mice remained diabetic. A leptin dose-response curve showed that subcutaneous leptin improved hyperglycemia and hyperinsulinemia in aP2-nSREBP-1c mice at doses that did not substantially alter hepatic steatosis or hepatic SCD enzymatic activity. Leptin treatment at this dose improved insulin-stimulated insulin receptor and insulin receptor substrate 2 (IRS-2) phosphorylation, IRS-2-associated PI3K activity, and Akt activity in liver. Together, these data suggest that CNS-mediated repression of SCD-1 contributes to leptin's antisteatotic actions. Intracerebroventricular leptin improves glucose homeostasis by improving insulin signal transduction in liver, but in this case the effect appears to be independent of SCD-1.

The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression

BackgroundIn humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo.MethodsWe constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15.ResultsThe effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene expression in the mice livers, which did not produce the therapeutic effects on alteration the lipid levels or the inhibition of atherosclerosis development. In contrast, the ribozyme RB15 RNA mediated by scAAV2-TTR-RB15 vector was expressed immediately at day-1 after transduction in HepG2 cells. The apoB mRNA levels were decreased 47% (p = 0.001), compared to the control vector scAAV2-TTR-RB15-mutant.ConclusionThis study provided evidence that the rAAV2 single-strand vector mediated a prolonged but not efficient transduction in mouse liver. However, the scAAV2 double-strand vector mediated a rapid and efficient gene expression in liver cells. This strategy using scAAV2 vectors represents a better approach to express small molecules such as ribozyme.

Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo.

An important limitation of recombinant adeno-associated virus (rAAV) vector efficiency is the requirement of hostcell-mediated synthesis of double-stranded DNA from the single-stranded genome. We have bypassed this step in a specialized self-complementary rAAV (scAAV) vector, by utilizing the tendency of AAV to package DNA dimers when the replicating genome is half the length of the wild type (wt). To produce these vectors efficiently, we have deleted the terminal resolution site (trs) from one rAAV TR, preventing the initiation of replication at the mutated end. These constructs generate single-stranded, inverted repeat genomes, with a wt TR at each end, and a mutated TR in the middle. After uncoating, the viral DNA folds through intramolecular base pairing within the mutant TR, which then proceeds through the genome to form a double-stranded molecule. We have used the scAAV to investigate barriers to rAAV transduction in the mouse liver, muscle and brain. In each tissue, scAAV was characterized by faster onset of gene expression and higher transduction efficiency. This study confirms earlier predictions that complementary-strand DNA synthesis is the primary barrier to rAAV-2 transduction. The scAAV is unaffected by this barrier, and provides an extremely efficient vector for gene transfer into many types of cells in vivo.

Sex significantly influences transduction of murine liver by recombinant adeno-associated viral vectors through an androgen-dependent pathway

AbstractA systematic evaluation of the influence of sex on transduction by recombinant adeno-associated viral vector (rAAV) indicated that transgene expression after liver-targeted delivery of vector particles was between 5- to 13-fold higher in male mice compared with female mice, irrespective of the proviral promoter or cDNA and mouse strain. Molecular analysis revealed that the rAAV genome was stably retained in male liver at levels that were 7-fold higher than those observed in females. Further, the sex difference in transduction was observed with AAV-2– and AAV-5–based vectors, which use distinct receptor complexes for infection. In concordance with the differences in AAV transduction, gel shift analysis with nuclear extracts derived from the liver of mice and humans revealed substantially higher binding of host nuclear protein to the rep-binding site (RBS) of AAV inverted terminal repeat (ITR) in males compared with females. Transduction efficiency and binding of nuclear protein to RBS was dramatically reduced in male mice by castration. In contrast, although oophorectomy did not significantly influence rAAV transduction, administration of 5α dihydrotestosterone, prior to gene transfer, increased stable hepatocyte gene transfer in females to levels observed in male mice, implying that androgens significantly influence hepatocyte gene transfer. Interestingly, sex did not have a significant effect on AAV gene transfer into nonhepatic tissue, indicating that there are distinct tissue- and sex-specific differences in the mechanisms responsible for efficient transduction with this vector. These results have significant implications for gene therapy of autosomal and acquired disorders affecting the liver.

Protein-tyrosine phosphatase activity in human adipocytes is strongly correlated with insulin-stimulated glucose uptake and is a target of insulin-induced oxidative inhibition

Protein-tyrosine phosphatases (PTPases), in particular PTP1B, have been shown to modulate insulin signal transduction in liver and skeletal muscle in animal models; however, their role in human adipose tissue remains unclear. The uptake of 14C-D-glucose in response to 10 or 100 nmol/L insulin was measured in isolated subcutaneous adipocytes from subjects with a mean age of 44 years (range, 26 to 58) and mean body mass index (BMI) of 35.6 (range, 29.7 to 45.5). The endogenous activity of total PTPases and specifically of PTP1B in immunoprecipitates was measured in cell lysates under an inert atmosphere with and without added reducing agents. Using nonlinear regression analysis, higher BMI was significantly correlated with lower adipocyte glucose uptake (r = 0.73, P = .01) and with increased endogenous total PTPase activity (r = 0.64, P = .04). Correlation with waist circumference gave similar results. The endogenous total PTPase activity also strongly correlated with insulin-stimulated glucose uptake (R = .89, P < .0001); however, the activity of PTP1B was unrelated to the level of glucose uptake. Consistent with the insulin-stimulated oxidative inhibition of thiol-dependent PTPases reported for 3T3-L1 adipocytes and hepatoma cells, treatment of human adipocytes with 100 nmol/L insulin for 5 minutes lowered endogenous PTPase activity to 37% of control (P < .001), which was increased 25% by subsequent treatment with dithiothreitol in vitro. Cellular treatment with diphenyleneiodonium (DPI), an NADPH oxidase inhibitor that blocks the cellular generation of H2O2 and reduces the insulin-induced reduction of cellular PTPase activity, also diminished insulin-stimulated glucose uptake by 82% (P = .001). These data suggest that total cellular PTPase activity, but not the activity of PTP1B, is higher in more obese subjects and is negatively associated with insulin-stimulated glucose transport. The insulin-stimulated oxidative inhibition of PTPases may also have an important permissive role in the transmission of the insulin signal to glucose transport in human adipocytes.

31. An Androgen Dependent Pathway Is Responsible for Gender Specific Differences in Transduction of Murine Liver by Recombinant Adeno-Associated Viral Vectors

31. An Androgen Dependent Pathway Is Responsible for Gender Specific Differences in Transduction of Murine Liver by Recombinant Adeno-Associated Viral Vectors

Subgroup B and F fiber chimeras eliminate normal adenovirus type 5 vector transduction in vitro and in vivo.

Altering adenovirus vector (Ad vector) targeting is an important goal for a variety of gene therapy applications and involves eliminating or reducing the normal tropism of a vector and retargeting through a distinct receptor-ligand pathway. The first step of Ad vector infection is high-affinity binding to a target cellular receptor. For the majority of adenoviruses and Ad vectors, the fiber capsid protein serves this purpose, binding to the coxsackievirus and adenovirus receptor (CAR) present on a variety of cell types. In this study we have explored a novel approach to altering Ad type 5 (Ad5) vector targeting based on serotypic differences in fiber function. The subgroup B viruses bind to an unidentified receptor that is distinct from CAR. The subgroup F viruses are the only adenoviruses that express two distinct terminal exons encoding fiber open reading frames. We have constructed chimeric fiber adenoviruses that utilize the tandem fiber arrangement of the subgroup F genome configuration. By taking advantage of serotypic differences in fiber expression, fiber shaft length, and fiber binding efficiency, we have developed a tandem fiber vector that has low binding efficiency for the known fiber binding sites, does not rely on an Ad5-based fiber, and can be grown to high titer using conventional cell lines. Importantly, when characterizing these vectors in vivo, we find the subgroup B system and our optimal tandem fiber system demonstrate reduced liver transduction by over 2 logs compared to an Ad5 fiber vector. These attributes make the tandem fiber vector a useful alternative to conventional strategies for fiber manipulation of adenovirus vectors.

Multiple muscles in the AMD quail can be "cross-corrected" of pathologic glycogen accumulation after intravenous injection of an [E1-, polymerase-] adenovirus vector encoding human acid-alpha-glucosidase.

Previously, in murine models of acid maltase deficiency (AMD), we demonstrated that intravenous administration of an improved adenovirus (Ad) vector encoding human acid alpha glucosidase (hGAA) resulted in liver transduction, followed by high-level hepatocyte-mediated secretion of hGAA into the plasma space. The hGAA secreted by the liver was taken up and targeted to muscle cell lysosomes. The levels of hGAA achieved by this approach resulted in clearance of lysosomal glycogen accumulations; in some muscle tissues the effect was prolonged (>6 months). We next wished to demonstrate whether this approach could be generalized across divergent species. To accomplish this goal, we determined whether a similar approach would also result in efficacy, but in a quail model of AMD. An [E1-, E2b-]Ad vector encoding hGAA was intravenously injected into AMD quails. At several time points thereafter, plasma, liver, and multiple muscle tissues were assayed for evidence of hGAA gene expression, liver-mediated hGAA secretion, uptake of hGAA by skeletal muscles, and evidence of glycogen correction in AMD skeletal muscles. These results were compared with those obtained from mock-injected AMD or wild-type quails. Intravenous [E1-, E2b-]Ad/hGAA vector injection resulted in high-level liver transduction and hepatic secretion of precursor forms of hGAA. The hepatically secreted hGAA was found to not only be efficiently taken up by cardiac and skeletal muscles, but was also proteolytically cleaved and processed equivalently to the quail-GAA protein detected in wild-type quails. The observations suggest that the signals regulating muscle cell uptake (but not proteolytic cleavage) of lysosomal enzymes are conserved and recognized across divergent species of vertebrates. Importantly, once localized to skeletal muscle lysosomes, the hGAA was able to effectively clear the glycogen accumulations present in AMD quail muscles. Adenovirus-mediated transduction of the hGAA gene, followed by hepatic secretion, uptake, and cross-correction of the pathologic glycogen accumulation noted in multiple muscles of both the AMD mouse and AMD quail, adds support to the notion that gene transfer strategies (Ad-mediated or other agents) targeting liver tissues with the hGAA gene are likely to be highly efficacious in humans affected by AMD.

A limited number of transducible hepatocytes restricts a wide-range linear vector dose response in recombinant adeno-associated virus-mediated liver transduction.

Recombinant adeno-associated virus (rAAV) vectors are promising vehicles for achieving stable liver transduction in vivo. However, the mechanisms of liver transduction are not fully understood, and furthermore, the relationships between rAAV dose and levels of transgene expression, total number of hepatocytes transduced, and proportion of integrated vector genomes have not been well established. To begin to elucidate the liver transduction dose response with rAAV vectors, we injected mice with two different human factor IX or Escherichia coli lacZ-expressing AAV serotype 2-based vectors at doses ranging between 4.0 x 10(8) and 1.1 x 10(13) vector genomes (vg)/mouse, in three- to sixfold increments. A 2-log-range linear dose-response curve of transgene expression was obtained from 3.7 x 10(9) to 3.0 x 10(11) vg/mouse. Vector doses above 3.0 x 10(11) vg/mouse resulted in disproportionately smaller increases in both the number of transduced hepatocytes and levels of transgene expression, followed by saturation at doses above 1.8 x 10(12) vg/mouse. In contrast, a linear increase in the number of vector genomes per hepatocyte was observed up to 1.8 x 10(12) vg/mouse concomitantly with enhanced vector genome concatemerization, while the proportion of integrated vector genomes was independent of the vector dose. Thus, the mechanisms that restrict a wide-range linear dose response at high doses likely involve decreased functionality of vector genomes and restriction of transduction to fewer than 10% of total hepatocytes. Such information may be useful to determine appropriate vector doses for in vivo administration and provides further insights into the mechanisms of rAAV transduction in the liver.

Impact of preimmunization on adenoviral vector expression and toxicity in a subcutaneous mouse cancer model.

Immune responses against adenoviral vectors may influence the toxicity and therapeutic effectiveness of adenovirus-mediated gene transfer and may be a limiting factor in adenovirus-mediated gene therapy. The purpose of this study was to determine the effects of preimmunization on intratumoral adenoviral transduction and systemic spread. The hypothesis was that increased doses of adenoviral vectors could overcome local neutralization without added systemic toxicity. The level and duration of gene expression were assessed as a function of time and dose after intratumoral delivery of adenoviral vector (AdV) encoding the luciferase reporter gene (AdV-luc) in a subcutaneous mouse mammary tumor model. Preimmunization resulted in significantly decreased gene expression in tumor and normal tissues (P < 0.01). The decrease was significantly greater in liver than in tumor. Increased AdV doses could be used to overcome the intratumoral inhibition without a concomitant increase in liver transduction. However, preimmunized animals showed greater toxicity than nai;ve animals (P < 0.001). The preimmunized group developed histologic evidence of grade 2-3 hepatic toxicity and increases in the average values of hepatic enzymes. In addition, there was a significant increase in mortality (P < 0.01) in the preimmunized group (12 of 20 animals) compared with the naive group (3 of 20 animals). These findings suggest that although preimmunity can inhibit systemic expression from adenoviral vectors, at high vector doses it may potentiate hepatotoxicity.

Efficient transduction of liver and muscle after in utero injection of lentiviral vectors with different pseudotypes.

In this study we investigate the efficacy of lentiviral vectors of different pseudotypes for gene transfer to tissues of the preimmune fetus. BALB/c fetuses at 14-15 days' gestation received lentiviral vectors carrying the transgene lacZ under the control of the human cytomegalovirus (CMV) promoter by intramuscular (i.m.) or intrahepatic (i.h.) injection. We pseudotyped the lentiviral vectors with vesicular stomatitis virus (VSV-G), with Mokola virus, or with Ebola virus envelope glycoproteins. We harvested the pups at time points between 5 days and 9 months following injection and performed a detailed histologic assessment. The efficiency and distribution of transduction after in utero administration was highly dependent upon the route of administration and the pseudotype of vector used. Biodistribution studies showed widespread distribution of vector sequences in multiple tissues, albeit at very low levels, and transduced cells were found in significant numbers only in liver, heart, and muscle. Overall, VSV-G was the most efficient in transducing hepatocytes, whereas Mokola and Ebola were more efficient in transducing myocytes. Transduction of cardiomyocytes was observed after both i.m. and i.h. injection of all three vectors. Our findings of long-term transduction of skeletal myocytes and cardiomyocytes after in utero administration suggest a novel strategy for the treatment of congenital muscular dystrophies.

G120K-PEG, a human GH antagonist, decreases GH signal transduction in the liver of mice

After receptor binding, growth hormone (GH) induces GH receptors (GHR) dimerization and JAK2 is activated after its association with a dimerized GHR, stimulating the tyrosyl phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-2 and Shc proteins. G120K-PEG, a GH antagonist is produced by a mutation that blocks GH action by preventing the GHR dimerization. This study shows that the inhibitory effect of G120K-PEG was maximal with a GH:G120K-PEG ratio of 1:100, as no increase in JAK2 tyrosyl phosphorylation was observed with this dose of GH. When the dose of GH was increased and with a GH:G120K-PEG ratio of 1:10 some tyrosyl phosphorylation of JAK2 could be observed. Additionally, GH-induced IRS-1, IRS-2 and SHC tyrosyl phosphorylation was inhibited ∼50% at equimolar concentrations of the antagonist of GH and almost abolished with a GH:G120K-PEG ratio of 1:100. The results clearly show that G120K-PEG inhibits GH signal transduction in mouse liver.

987. Transduction of Rabbit Liver with Plasmid DNA Using Catheter-Based Procedures

987. Transduction of Rabbit Liver with Plasmid DNA Using Catheter-Based Procedures

Role of hepatocyte direct hyperplasia in lentivirus-mediated liver transduction in vivo.

Lentiviral vectors have been used for gene transfer into the liver, but the ability of these vectors to efficiently transduce quiescent hepatocytes remains controversial. Regardless, lentivirus-mediated gene transfer is greatly enhanced when delivered during hepatocellular cycling. For this reason, the present study was designed to determine the role of hepatocyte proliferation in the enhancement of lentiviral transduction by using three different modes of liver regeneration: (1) compensatory regeneration stimulated by two-thirds partial hepatectomy, (2) direct hyperplasia after intragastric administration of the primary mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), and (3) a combination of modes 1 and 2. Vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentiviral vector expressing beta-galactosidase was administered to mice via the peripheral circulation after a regeneration stimulus. Gene transfer as measured by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) staining showed 30-fold higher levels of liver transduction in groups 1 and 2 as compared with the non-liver-manipulated control group (p < 0.005). The combination of TCPOBOP and partial hepatectomy (group 3) resulted in an ~80-fold increase in transduction efficiency compared with the control animals. The enhanced transduction was consistent with higher levels of hepatocellular proliferation observed in animals that received both treatments compared with either single treatment alone. Importantly, the hepatocytes were the predominant cell type transduced, although transgene expression was observed in a low number of nonparenchymal cells regardless of which liver stimulus was received. Biodistribution studies confirmed that most of the gene transfer was limited to the liver and spleen. Taken together, this study suggests that disease-induced cellular proliferation in the liver will enhance the utility of this vector in treating diseases such as viral hepatitis, liver cirrhosis, and cancer.

Envelope-targeted retrovirus vectors transduce melanoma xenografts but not spleen or liver.

Many cancer gene therapy applications would benefit from the development of targeted vectors that could deliver genes in vivo. We have previously achieved efficient in vitro targeting of retrovirus vectors to melanoma cells by fusion of a single chain antibody recognizing the high-molecular-weight melanoma-associated antigen (HMWMAA), followed by a blocking peptide and a matrix metalloprotease cleavage site, to the amino terminus of the murine leukemia virus amphotropic strain envelope. Here we report that up to 3% of cells within an HMWMAA-positive tumor xenograft were infected following a single injection of targeted vector into the tumor and up to 10% of tumor cells became infected when they were co-injected with viral producer cells. No infected cells were detected after delivery of targeted vectors to HMWMAA-negative tumor xenografts. Intraperitoneal injection of amphotropic vectors or producer cells resulted in transduction in spleen and liver, which was not detected when targeted vectors or producer cells were used. Our results demonstrate the feasibility of using targeted retroviral vectors for in vivo gene delivery to tumors and highlight the safety benefits of targeted vectors that do not infect other host tissues.

Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity.

The serotypes of adeno-associated virus (AAV) have the potential to become important resources for clinical gene therapy. In an effort to compare the role of serotype-specific virion shells on vector transduction, we cloned each of the serotype capsid coding domains into a common vector backbone containing AAV type 2 replication genes. This strategy allowed the packaging of AAV2 inverted terminal repeat vectors into each serotype-specific virions. Each of these helper plasmids (pXR1 through pXR5) efficiently replicated the transgene DNA and expressed helper proteins at nearly equivalent levels. In this study, we observed a correlation between the amount of transgene replication and packaging efficiency. The physical titer of these hybrid vectors ranged between 1.3 x 10(11) and 9.8 x 10(12)/ml (types 1 and 2, respectively). Of the five serotype vectors, only types 2 and 3 were efficiently purified by heparin-Sepharose column chromatography, illustrating the high degree of similarity between these virions. We analyzed vector transduction in reference and mutant Chinese hamster ovary cells deficient in heparan sulfate proteoglycan and saw a correlation between transduction and heparan sulfate binding data. In this analysis, types 1 and 5 were most consistent in transduction efficiency across all cell lines tested. In vivo each serotype was ranked after comparison of transgene levels by using different routes of injection and strains of rodents. Overall, in this analysis, type 1 was superior for efficient transduction of liver and muscle, followed in order by types 5, 3, 2, and 4. Surprisingly, this order changed when vector was introduced into rat retina. Types 5 and 4 were most efficient, followed by type 1. These data established a hierarchy for efficient serotype-specific vector transduction depending on the target tissue. These data also strongly support the need for extending these analyses to additional animal models and human tissue. The development of these helper plasmids should facilitate direct comparisons of serotypes, as well as begin the standardization of production for further clinical development.