Frits J. Fallaux

Spacers increase the accessibility of peptide ligands linked to the carboxyl terminus of adenovirus minor capsid protein IX.

ABSTRACT The efficiency and specificity of gene transfer with human adenovirus (hAd)-derived gene transfer vectors would be improved if the native viral tropism could be modified. Here, we demonstrate that the minor capsid protein IX (pIX), which is present in 240 copies in the Ad capsid, can be exploited as an anchor for heterologous polypeptides. Protein IX-deleted hAd5 vectors were propagated in hAd5 helper cells expressing pIX variants, with heterologous carboxyl-terminal extensions of up to 113 amino acids in length. The extensions evaluated consist of alpha-helical spacers up to 75 Å in length and to which peptide ligands were fused. The pIX variants were efficiently incorporated into the capsids of Ad particles. On intact particles, the MYC-tagged-pIX molecules were readily accessible to anti-MYC antibodies, as demonstrated by electron microscopic analyses of immunogold-labeled virus particles. The labeling efficiency improved with increasing spacer length, suggesting that the spacers lift and expose the ligand at the capsid surface. Furthermore, we found that the addition of an integrin-binding RGD motif to the pIX markedly stimulated the transduction of coxsackievirus group B and hAd receptor-deficient endothelioma cells, demonstrating the utility of pIX modification in gene transfer. Our data demonstrate that the minor capsid protein IX can be used as an anchor for the addition of polypeptide ligands to Ad particles.

Isolated-organ perfusion for local gene delivery: efficient adenovirus-mediated gene transfer into the liver

Targeted gene delivery is essential for gene therapy involving in vivo gene transfer. In the present study we analyzed the efficiency and tissue-specificity of gene transfer into the liver with recombinant adenoviruses. Adenovirus vectors carrying the E. coli lacZ gene (Ad.RSV.β-gal) and the firefly luciferase gene (AdCMV-luc) as reporters were administered to the liver of adult Wistar rats, either via infusion into the portal vein (intraportal infusion; IPI) or via perfusion of the vascularly isolated liver (isolated liver perfusion; ILP). Ex vivo liver perfusion experiments with Ad.RSV.β-gal were used to optimize the conditions for hepatic gene transfer. Ex vivo perfusion of rat livers with 2 × 109 plaque forming units (p.f.u.) Ad.RSV.β-gal was sufficient to infect about 20% of the liver parenchymal cells. Perfusion with chelating agents (1 mM EGTA, or 2 mM EDTA) prior to the administration of the vector increased the efficiency to at least 40%. Similar efficiencies were obtained in experiments with liver lobes of Rhesus monkeys. In vivo administration of AdCMV-luc via ILP resulted in a significantly more efficient (P = 0.028) and also more reproducible gene transfer when compared to IPI. Although detectable in both groups, extrahepatic luciferase expression was considerably reduced in the ILP group. Our data demonstrate that IPL can be used for efficient and reproducible liver-specific gene delivery. Therefore, we think that the perfusion of vascularly isolated organs is useful as a modality for the tissue-specific administration of recombinant adenovirus vectors.

The human clotting factor VIII cDNA contains an autonomously replicating sequence consensus- and matrix attachment region-like sequence that binds a nuclear factor, represses heterologous gene expression, and mediates the transcriptional effects of sodium butyrate.

Expression of the human blood-clotting factor VIII (FVIII) cDNA is hampered by the presence of sequences located in the coding region that repress transcription. We have previously identified a 305-bp fragment within the FVIII cDNA that is involved in the repression (R.C. Hoeben, F.J. Fallaux, S.J. Cramer, D.J.M. van den Wollenberg, H. van Ormondt, E. Briet, and A.J. van der Eb, Blood 85:2447-2454, 1995). Here, we show that this 305-bp region of FVIII cDNA contains sequences that resemble the yeast (Saccharomyces cerevisiae) autonomously replicating sequence consensus. Two of these DNA elements coincide with AT-rich sequences that are often found in matrix attachment regions or scaffold-attached regions. One of these elements, consisting of nucleotides 1569 to 1600 of the FVIII cDNA (nucleotide numbering is according to the system of Wood et al. (W.I. Wood, D.J. Capon, C.C. Simonsen, D.L. Eaton, J. Gitschier, D. Keyt, P.H. Seeburg, D.H. Smith, P. Hollingshead, K.L. Wion, et al., Nature [London] 312:330-337,1984), binds a nuclear factor in vitro but loses this capacity after four of its base pairs have been changed. A synthetic heptamer of this segment can repress the expression of a chloramphenicol acetyltransferase (CAT) reporter gene and also loses this capacity upon mutation. Furthermore, we demonstrate that repression by FVIII sequences can be relieved by sodium butyrate. We demonstrate that the synthetic heptamer (FVIII nucleotides 1569 to 1600), when placed upstream of the Moloney murine leukemia virus long terminal repeat promoter that drives the CAT reporter, can render the CAT reporter inducible by butyrate. This effect was absent when the same element was mutated. The stimulatory effect of butyrate could not be attributed to butyrate-responsive elements in the studied long terminal repeat promoters. Our data provide a functional characterization of the sequences that repress expression of the FVIII cDNA. These data also suggest a link between transcriptional repression by FVIII cDNA elements and the stimulatory effect of butyrate on FVIII cDNA expression.

Ammonium sulphate precipitation of recombinant adenovirus from culture medium: an easy method to increase the total virus yield

In the majority of the methods for purifying and concentrating recombinant adenoviruses (rAds) the virus that is associated with the helper cells is harvested, while the virus that is present in the cell-culture medium is discarded. During routine propagation of adenovirus type-5 vectors at optimised conditions we noted that, on average, 47% of the total amount of virus is present in the culture medium. To recover and concentrate these rAds from the medium, we devised a method, which is based on ammonium sulphate ((NH4)2SO4) precipitation. At 40% (NH4)2SO4 saturation, 95 ± 6% of the available virus precipitates from the medium, while the majority of the protein (85%) remains in solution. In contrast to adenovirus precipitation with polyethylene glycol, the (NH4)2SO4precipitation technique allows collection of precipitated rAds by filtration. We demonstrate here that (NH4)2SO4 precipitation of rAds from cell-culture medium is a simple and fast technique that can be used in combination with standard virus isolation methods to increase the yields of rAds.

Gene therapy for the hemophilias.

This review discusses the progress of gene therapy for the hemophilias. The development of gene therapy for hemophilia A has been more problematic than that for hemophilia B. It is now well established that reduced expression of the human clotting factor VIII cDNA is caused by transcriptional repression. Multiple sequences within the factor VIII cDNA are involved. So far, attempts to improve the factor VIII cDNA expression have been unsuccessful. However, improved retroviral vectors and adenovirus‐based vectors have been constructed that increase factor VIII expression. The use of these vectors has resulted in clinically relevant levels of human factor VIII in mice and hemophilic dogs. Thus, gene therapy for hemophilia A has reached the same developmental stage as that for hemophilia B. If further improvements can increase the persistence of expression and decrease the immunologic responses, phase I clinical trials in human individuals can be considered.

Apolipoprotein E2 (Lys146→Gln) Causes Hypertriglyceridemia due to an Apolipoprotein E Variant–Specific Inhibition of Lipolysis of Very Low Density Lipoproteins–Triglycerides

The apolipoprotein E2 (Lys146-->Gln) variant is associated with a dominant form of familial dysbetalipoproteinemia. Heterozygous carriers of this variant have elevated levels of plasma triglycerides, cholesterol, and apolipoprotein E (apoE). It was hypothesized that the high amounts of triglycerides in the very low density lipoprotein (VLDL) fraction are due to a disturbed lipolysis of VLDL. To test this hypothesis, apoE knockout mice were injected with an adenovirus containing the human APOE*2 (Lys146-->Gln) gene, Ad-E2(146), under the control of the cytomegalovirus promoter. ApoE knockout mice injected with an adenovirus vector encoding human apoE3 (Ad-E3) were used as controls. Five days after adenovirus injection, plasma cholesterol levels of mice injected with a high dose of Ad-E2(146) (2x10(9) plaque-forming units) were not changed compared with preinjection levels, whereas in the group who received a low dose of Ad-E2(146) (5x10(8) plaque-forming units) and in the groups injected with a low or a high dose of Ad-E3, plasma cholesterol levels were decreased 5-, 6-, and 12-fold, respectively. Plasma triglycerides were not affected in mice injected with Ad-E3. In contrast, a 7-fold increase in plasma triglycerides was observed in mice injected with the low dose of Ad-E2(146) compared with mice injected with Ad-E3. Injection with the high dose of Ad-E2(146) resulted in a dramatic increase of plasma triglycerides (50-fold compared with Ad-E3 injection). In vitro lipolysis experiments showed that the lipolysis rate of VLDLs containing normal amounts of apoE2 (Lys146-->Gln) was decreased by 54% compared with that of VLDLs containing comparable amounts of apoE3. The in vivo VLDL-triglyceride production rate of Ad-E2(146)-injected mice was not significantly different from that of Ad-E3-injected mice. These results demonstrate that expression of apoE2 (Lys146-->Gln) causes hypertriglyceridemia due to an apoE variant-specific inhibition of the hydrolysis of VLDL-triglycerides.

Efficient in vivo knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA

BackgroundAdenovirus (Ad) mediated gene transfer is a well-established tool to transiently express constructs in livers of mice in vivo. In the present study, we determined the specificity and efficiency of Ad vectors expressing short hairpin (sh) RNA constructs to knock-down the estrogen receptor α (ERα).ResultsTwo different shRNA constructs derived from the murine ERα coding sequence were designed (shERα). In vitro, transfection of three mouse cell lines with pSUPER-shERα constructs resulted in up to 80% reduction of endogenous ERα activity. A single mismatch in the target sequence eliminated the reduction of ERα activity, demonstrating the specificity of shERα. The subsequently generated Ad.shERα vectors were equally effective in vitro. In vivo, intravenous administration of Ad.shERα resulted in 70% reduced hepatic mouse ERα mRNA levels. Co-injection of Ad.shERα with an Ad vector containing a luciferase (luc) gene driven by an estrogen responsive element (ERE) containing promoter resulted in a significant (90% on day five) down-regulation of hepatic luciferase activity, as determined by non-invasive optical imaging. Down-regulation was sustained up to day seven post-injection.ConclusionAd mediated transfer of shERα expression constructs results in efficient and specific knockdown of endogenous ERα transcription both in vitro and in vivo.

Adenovirus-Mediated Gene Transfer

Recombinant adenoviruses are attractive vectors for short-term expression in mouse liver and primary cell lines. Various versatile vector systems have been developed which can be used for the reliable production of recombinant adenoviruses. This protocol describes the entire process for the production of recombinant adenoviruses using the AdEasy system. This protocol will give a practical step-by-step description from the cloning of the gene of interest until the in vivo administration in mice. The entire process will take about 8 weeks to complete.