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Adeno-associated virus (AAV) vectors have shown remarkable efficiency for gene delivery to cultured cells and in animal models of human disease. However, limitations to AAV vectored gene transfer exist after intravenous transfer, including off-target gene delivery (e.g., liver) and low transduction of target tissue. Here, we show that during production, a fraction of AAV vectors are associated with microvesicles/exosomes, termed vexosomes (vector-exosomes). AAV capsids associated with the surface and in the interior of microvesicles were visualized using electron microscopy. In cultured cells, vexosomes outperformed conventionally purified AAV vectors in transduction efficiency. We found that purified vexosomes were more resistant to a neutralizing anti-AAV antibody compared to conventionally purified AAV. Finally, we show that vexosomes bound to magnetic beads can be attracted to a magnetized area in cultured cells. Vexosomes represent a unique entity which offers a promising strategy to improve gene delivery.

Most clinical protocols involving adenovirus (Ad) vectors for gene therapy use a vector based on serotype 5 (Ad5). We believe that this serotype is not suitable for all gene therapy applications and that alternative vectors based on other serotypes should be developed. We have compared the ability of Ad5, Ad11p, Ad16p, and a chimpanzee Ad (CV23) to infect human low-passage brain tumor cells as well as primary glioma cells sorted into a CD133+ and CD133– population. Cancer stem cells have been shown to reside in the CD133+ population of cells in human glioma tumors and they are of considerable interest in glioma therapy. Ad16p and CV23 infected the low-passage brain tumor cell lines and also the CD133+ and CD133– primary tumor cells most efficiently. Interestingly, as the passage number of the cells increased, the infection capacity of Ad5 increased significantly, whereas this was not seen for CV23. To ensure the therapeutic effect of Ad vectors on brain tumors, the vector must be capable of addressing both the CD133+ cancer stem cells and the CD133– cells of the tumor. In particular, Ad16 and CV23 are meeting this challenge.

Top of pageAbstract Background: Adenoviruses of species A, B, C, D, E and F differ in tropism. In order to address the molecular determinants of adenovirus tropism the distribution of adenovirus receptors on cells of hematopoietic and endothelial origin, and the expression in these cells was assessed. Results: The receptors for species B adenoviruses were most abundantly expressed. CAR binding amino acid residues are not conserved in their fiber knobs consequently CAR can not be used as a receptor. Virions of Ad11p, Ad35 & Ad3 (species B) showed high- affinity to CD34(+) cells, Ad7, Ad11a (species B) and Ad37 (species D) showed intermediate affinity whereas Ad4 (species E), Ad5 (species C), Ad31 (species A) and Ad41 (species F) hardly bound to CD34 hematopoietic progenitor cells. Only species B and species D adenovirus were expressed in these cells. Based on DNA homology two groups are defined within species B. Ad3, 7, 16, 21 (B:1), and Ad11, 14, 34, 35 and 50 (B:2). Ad3 and Ad7 cause acute respiratory tract infections, whereas Ad11, 34 and 35 cause persistent infections of the urinary tract. We suggest two different receptors for species B adenoviruses: sBAR common to species B members and sB2AR unique for Ad11 and Ad35 members of species B:2. Ad11 virions blocked the binding of Ad3 or Ad7 species B to 70%, whereas Ad3 blocked the binding of Ad11 and Ad35 only partially 30%. sBAR is sensitive to trypsin and EDTA treatment of A549 cells or J82 cells whereas sB2AR is resistent. The binding of Ad3 and Ad7 was restored with Ca++. Non permissive chinese hamster ovary (CHO) cells were transfected with the BC1 or BC2 isoforms of CD46 |[ndash]| cDNA. Ad11 virions did but Ad5 virions did not bind to CD46 transfected CHO cells. Adenovirus 11 and 35 can attach and infect primary lymphocytes and monocytes but hexon expression in T- cells required prior activation, whereas neither Ad3, Ad7 nor Ad5 were expressed in the primary PBMCs. Assessment of the transduction ability of established hematopoietic cell lines by a species B vector revealed that Jurkat, U937 and K562 were more efficiently transduced than DG75 cells. EKC causing Ad37 (species D) uses 2-3 linked sialic acid as a primarily functional receptor. The structure of the fiber knob of Ad37 and Ad19p was resolved at 1.5-2|[Aring]| by W. Burmeister using X-ray crystallography. The binding site for sialyl lactose was identified on the top of the knob close to the 3-fold axis of the trimer. NB Ad37 infects CD34+ cells Conclusion: Species B and D adenovirus using CD46 and 2-3- linked sialic acid as receptors, respectively, infected hematopoietic progenitor cells. Ad11 and Ad35 were more efficient than all other assessed adenoviruses.

Most clinical protocols using adenovirus vectors for gene therapy are using a vector based on serotype 5 (Ad5). However, we believe that this vector is not suitable for all needs of gene therapy and alternative vectors based on other serotypes should be developed. We have analysed the capacity of a chimpanzee adenovirus to infect human low passage glioma cells from seven different donors. Even though this is a non-human virus, it infected the human cells very efficiently and was superior to Ad5 on low passage cells from all donors except one where Ad5 and the chimpanzee adenovirus were on par. When the glioma cells were passaged to a higher passage number, the infection capacity for Ad5 increased significantly on all cells tested except one, whereas it was constant or the reverse effect for the chimpanzee adenovirus. We show that the receptor is different from the previously known adenovirus receptors CAR and CD46 and not dependent of N- and O-linked glycosylation or glycosaminoglycans. An extraordinary high binding capacity to all cells tested was seen for this virus in the presence of fetal bovine serum, but not human serum. Further studies showed that bovine serum albumin, but not human serum albumin was responsible for this effect.