Inhibitors of Angiogenesis.

Angiogenesis plays a pivotal role in malignant, ischemic, inflammatory, infectious and immune disorders. The increasing molecular understanding of angiogenic processes fostered the development of strategies to induce or inhibit angiogenesis for therapeutic purposes. Here, we focus on anti-angiogenic therapies, which represent a standard of care in the treatment of different cancer types and in neovascular age-related macular degeneration. Specifically, strategies related to the blockade of angiogenic proteins and receptors will be outlined covering both preclinical and clinical aspects. Finally, examples of gene therapy based anti-angiogenic approaches are presented.

Successful target cell transduction of capsid-engineered rAAV vectors requires clathrin-dependent endocytosis.

Cell surface targeting of recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to modify AAV's natural tropism. As modification of the capsid surface is likely to affect the mechanism of vector internalization and consequently the vector's intracellular fate, we investigated early steps in cell transduction of rAAV capsid insertion mutants. Mutants displaying peptides with neutral overall charge at position 587 transduced cells independently of AAV2's primary receptor heparan sulfate proteoglycan (HSPG), whereas mutants carrying positively charged insertions were capable of HSPG binding with affinities correlating with their net positive charge. Whereas rAAV2 is internalized via an HSPG- and clathrin-dependent pathway, HSPG-binding mutants used a clathrin- and caveolin-independent mechanism. Surprisingly, although this pathway was as efficient in mediating vector entry as the one used by rAAV2, successful cell transduction was hampered at a post-entry step, presumably caused by inefficient endosomal escape. In contrast, HSPG-independent, clathrin-dependent internalization used by non-HSPG-binding mutants correlated with efficient nuclear delivery of vector genomes and robust transgene expression. These findings indicate that cell surface targeting strategies should direct uptake of rAAV targeting vectors to clathrin-mediated endocytosis, the naturally evolved entry route of AAV, to promote successful intracellular processing and re-targeting of rAAV's tropism.

Real-time single-molecule imaging of the infection pathway of an adeno-associated virus.

We describe a method, based on single-molecule imaging, that allows the real-time visualization of the infection pathway of single viruses in living cells, each labeled with only one fluorescent dye molecule. The tracking of single viruses removes ensemble averaging. Diffusion trajectories with high spatial and time resolution show various modes of motion of adeno-associated viruses (AAV) during their infection pathway into living HeLa cells: (i) consecutive virus touching at the cell surface and fast endocytosis; (ii) free and anomalous diffusion of the endosome and the virus in the cytoplasm and the nucleus; and (iii) directed motion by motor proteins in the cytoplasm and in nuclear tubular structures. The real-time visualization of the infection pathway of single AAVs shows a much faster infection than was generally observed so far.

Capsid Engineering Overcomes Barriers Toward Adeno-Associated Virus Vector-Mediated Transduction of Endothelial Cells.

Endothelial cells (EC) are targets in gene therapy and regenerative medicine, but they are inefficiently transduced with adeno-associated virus (AAV) vectors of various serotypes. To identify barriers hampering efficient transduction and to develop an optimized AAV variant for EC transduction, we screened an AAV serotype 2-based peptide display library on primary human macrovascular EC. Using a new high-throughput selection and monitoring protocol, we identified a capsid variant, AAV-VEC, which outperformed the parental serotype as well as first-generation targeting vectors in EC transduction. AAV vector uptake was improved, resulting in significantly higher transgene expression levels from single-stranded vector genomes detectable within a few hours post-transduction. Notably, AAV-VEC transduced not only proliferating EC but also quiescent EC, although higher particle-per-cell ratios had to be applied. Also, induced pluripotent stem cell-derived endothelial progenitor cells, a novel tool in regenerative medicine and gene therapy, were highly susceptible toward AAV-VEC transduction. Thus, overcoming barriers by capsid engineering significantly expands the AAV tool kit for a wide range of applications targeting EC.

Engineering adeno-associated virus 2 vectors for targeted gene delivery to atherosclerotic lesions.

Targeted delivery of biological agents to atherosclerotic plaques may provide a novel treatment and/or useful tool for imaging of atherosclerosis in vivo. However, there are no known viral vectors that possess the desired tropism. Two plaque-targeting peptides, CAPGPSKSC (CAP) and CNHRYMQMC (CNH) were inserted into the capsid of adeno-associated virus 2 (AAV2) to assess vector retargeting. AAV2-CNH produced significantly higher levels of transduction than unmodified AAV2 in human, murine and rat endothelial cells, whereas transduction of nontarget HeLa cells was unaltered. Transduction studies and surface plasmon resonance suggest that AAV2-CNH uses membrane type 1 matrix metalloproteinase as a surface receptor. AAV2-CAP only produced higher levels of transduction in rat endothelial cells, possibly because the virus was found to be affected by proteasomal degradation. In vivo substantially higher levels of both peptide-modified AAV2 vectors was detected in the brachiocephalic artery (site of advanced atherosclerotic plaques) and aorta, whereas reduced levels were detected in all other organs examined. These results suggest that in the AAV2 platform the peptides are exposed on the capsid surface in a way that enables efficient receptor binding and so creates effective atherosclerotic plaque targeted vectors.

AAV-mediated gene transfer to cardiac cells in a heterotopic rat heart transplantation model.

INTRODUCTION: Adeno-associated virus (AAV) vectors offer the possibility to transfer genes to a wide range of organ and cell types. To determine the efficiency of AAV-mediated gene transfer to cardiac cells, vectors were administered to the heart under various conditions. METHODS: In Sprague-Dawley rats, AAV vectors based on serotype 2 and coding for beta-galactosidase were injected via coronaries into hypothermic nonbeating and normothermic beating hearts before transplantation. In addition, vectors were injected intravenously or into the thigh muscle. After 28 days all animals were humanely killed and organs explanted for analysis. RESULTS: Transgenic DNA was always detectable in the liver and the heart, irrespective of the application mode. However, transgenic mRNA could not be determined in the transplanted hearts. In contrast, direct injection into the thigh muscle resulted in transgenic mRNA production and marker gene expression. After systemic application, transgenic mRNA was detected in the liver but not in the heart. CONCLUSION: The results of our study indicated that AAV-mediated gene transfer to cardiac cells is possible. However, it was impossible to detect transgenic mRNA or marker gene expression in the transplanted hearts after intracoronary perfusion or systemic injection.

Bis[platinum(II)] and bis[platinum(IV)] complexes with optically active bis(vicinal-1,2-diamines) and their interaction with DNA.

Bis[platinum(II)] [Cl2Pt(LL)PtCl2] complexes 2,5 and 8 with chiral non-racemic ligands: 1a-c (LL = (R,R), (S,S) and (R,S) N,N'-bis(3,4-diaminobutyl)hexanediamide); 4a,b (LL = (R,R) and (S,S) N,N'-bis[3,4-bis(diaminobutyl)] urea); 7a-d (LL' = (R,R), (S,S), (R,S) and (S,R) 4,5-diamino-N-(3,4-diaminobutyl) pentanamide) and bis[platinum(IV)] complex 10-13 with ligands 1a,b and 4a,b have been prepared and characterized by IR, 1H, 13C and 195Pt NMR spectra. The interactions of 2a-c, 5a, 5b, 8a-d and 10a with dsDNA were investigated with the goal of examining whether the chirality, the nature of the spacer and the oxidation state have an influence on platinum-DNA binding properties. All the bis[platinum(II)] complexes form with dsDNA intra- and interstrand crosslinks and crosslinks over sticky ends, whereas the bis[platinum(IV)] complex 10a only forms intra- and interstrand crosslinks. The platinum-DNA coordination sites were determined by the T4 DNA polymerase footprinting method. The results show that all investigated bis(platinum) complexes have high preference towards distinct purines. All isomeric bis(amide) 2a-c and mono(amide) 8a-d complexes exhibit nearly the same binding pattern, whereas the ureide complexes 5a and 5b have other coordination sites with higher sequence preference. Interestingly, the ureides 5a and 5b differ in their coordination sites not only in comparison to the bis(amides) 2a-c and mono(amides) 8a-d, but also between each other. The bis[platinum(IV)] complex 10a also differs in coordination sites in comparison to all the bis[platinum(II)] compounds.

A novel directed evolution method to enhance cell-type specificity of adeno-associated virus vectors.

Clinical application of viral vectors is often hampered by the lack of selectivity of viral particles for the targeted tissue. This drawback decreases the efficiency of gene delivery and raises safety concerns. We successfully established a novel in vitro evolution protocol to engineer adeno-associated virus vectors with increased selectivity for designated target cells. Subjecting a peptide-display library of AAV capsids to negative selection cycles on human primary fibroblasts and to positive selection cycles on a human melanoma cell line, we isolated several variants with up to 3.7-fold increased specificity for malignant cells in comparison to fibroblasts and other cell types. These mutants can be used to achieve high levels of gene transfer to target cells reducing undesired transduction of neighbouring tissues.

Molecular interaction of DNA with bisplatinum(II) complexes having bis(vicinal 1,2-diamines) as ligand.

The interaction of novel, tetrafunctional bisplatinum compounds with DNA was investigated. These compounds have bis(vicinal 1,2-diamines) as ligand. The reactions' efficiency, types of cross-links, alterations of the global DNA structure, and sequence selectivity differ significantly from the corresponding features of cisplatin. In particular, they form multiple complexes with dsDNA, which include intrastrand, interstrand and interhelical cross-links and cross-links over sticky ends. The novel compounds are able to untwist but not shorten dsDNA. The reactivity and adduct-forming efficiency of these compounds is, depending on the spacer length, 100-200-fold higher than that of cisplatin. As a consequence, interstrand cross-links are also formed to a higher extent. The chemical stability of the interstrand cross-links against cyanide ions, however, is weaker than that of interstrand cross-links formed by cisplatin, suggesting that each platinum sphere of a bisplatinum compound forms intrastrand cross-links. With dsDNA, they show a preference toward purines, particularly guanines, but they apparently are also coordinated to other nucleobases. Their sequence selectivity toward dsDNA is higher than that of cisplatin. Thus, the novel compounds extend the spectrum of alternative platinum-based compounds with chemical features different from cisplatin.

Genetic modifications of the adeno-associated virus type 2 capsid reduce the affinity and the neutralizing effects of human serum antibodies.

The high prevalence of human serum antibodies against adeno-associated virus type 2 (AAV) vectors represents a potential limitation for in vivo applications. Consequently, the development of AAV vectors able to escape antibody binding and neutralization is of importance. To identify capsid domains which contain major immunogenic epitopes, six AAV capsid mutants carrying peptide insertions in surface exposed loop regions (I-261, I-381, I-447, I-534, I-573, I-587) were analyzed. Two of these mutants, I-534 and I-573, showed an up to 70% reduced affinity for AAV antibodies as compared to wild-type AAV in the majority of serum samples. In addition, AAV mutant I-587 but not wild-type AAV efficiently transduced cells despite the presence of neutralizing antisera. Taken together, the results show that major neutralizing effects of human AAV antisera might be overcome by the use of AAV capsid mutants.

Engagement of the B-cell antigen receptor (BCR) allows efficient transduction of ZAP-70-positive primary B-CLL cells by recombinant adeno-associated virus (rAAV) vectors.

Engagement of the B-cell antigen receptor (BCR) by crosslinking of the surface immunoglobulin (sIg) homodimer was studied for recombinant adeno-associated virus (rAAV)-mediated gene transfer into B-cell chronic lymphocytic leukaemia (B-CLL) cells. Leukemic cells obtained from 20 patients were stimulated with anti-sIg-directed antibodies and transduced with rAAV vectors coding for enhanced green fluorescent protein (EGFP) (AAV/EGFP) or CD40L (AAV/CD40L). Transduction of B-CLL cells was enhanced after BCR engagement compared to unstimulated controls (P=0.0356). BCR crosslinking induced a significant, dose- and time-dependent upregulation of heparan sulfate proteoglycan (HSPG), the primary receptor for AAV, on B-CLL cells (mean: 38.2 versus 1.7%; P=0.0006). A correlation of HSPG expression after BCR crosslinking with transduction efficiency by AAV/EGFP (P=0.0153) and AAV/CD40L (P=0.0347) was observed. High expression of zeta-associated protein 70 (ZAP-70) in B-CLL cells correlated with a better transduction efficiency by AAV/EGFP (P<0.0001) and AAV/CD40L (P=0.002), respectively: 48 h after transduction of ZAP-70-positive samples, transgene expression was seen in a mean of 33.8% (s.e.m. 3.7%) and 28.9% (s.e.m. 6.7%) of cells, respectively, and could be specifically blocked by heparin, a soluble competitor of HSPG (P<0.0001). In summary, engagement of the BCR on ZAP-70 positive B-CLL cells allows efficient rAAV-mediated gene delivery.

Receptor targeting of adeno-associated virus vectors.

Adeno-associated virus (AAV) is a promising vector for human somatic gene therapy. However, its broad host range is a disadvantage for in vivo gene therapy, because it does not allow the selective tissue- or organ-restricted transduction required to enhance the safety and efficiency of the gene transfer. Therefore, increasing efforts are being made to target AAV-2-based vectors to specific receptors. The studies summarized in this review show that it is possible to target AAV-2 to a specific cell. So far, the most promising approach is the genetic modification of the viral capsid. However, the currently available AAV-2 targeting vectors need to be improved with regard to the elimination of the wild-type AAV-2 tropism and the improvement of infectious titers. The creation of highly efficient AAV-2 targeting vectors will also require a better understanding of the transmembrane and intracellular processing of this virus.

Standard heparin, low molecular weight heparin, low molecular weight heparinoid, and recombinant hirudin differ in their ability to inhibit transduction by recombinant adeno-associated virus type 2 vectors.

Recombinant adeno-associated virus type 2 (rAAV) is a promising vector for in vivo gene therapy. Transduction by rAAV requires binding to heparan sulfate proteoglycan on the cell surface, and heparin can block this binding. Because heparin is administered to most patients undergoing cardiovascular gene transfer in order to prevent thrombotic events, it is important to identify anticoagulants which do not interfere with rAAV transduction. Therefore, we examined the influence of different anticoagulants on rAAV transduction in vitro. rAAV transduction was inhibited by 40.5 +/- 7.9% at heparin concentrations of 0.1 U/ml, and by 81.7 +/- 3.6% at 1.0 U/ml. The low molecular weight (LMW) heparin tinzaparin inhibited rAAV transduction by 20.2 +/- 3.8% at 0.1 U/ml and 37.1 +/- 1.8% at 1.0 U/ml. The inhibitory effect was significantly weaker compared with heparin at 1.0 U/ml, (P < 0.01). The LMW heparinoid danaparoid inhibited rAAV transduction by 8.8 +/- 3.5% at 0.1 U/ml (P < 0.01 compared with heparin). In contrast, recombinant hirudin did not interfere at all with rAAV transduction. In summary, the results demonstrate that inhibition of rAAV transduction by heparin occurs rapidly and at therapeutically used concentrations. LMW heparinoids and above all recombinant hirudin might be alternatives for heparin when vascular gene transfer with rAAV requires transient anticoagulation.