Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors

Abstract

We describe receptor-targeted adeno-associated viral (AAV) vectors that allow genetic modification of rare cell types ex vivo and in vivo while showing no detectable off-targeting. Displaying designed ankyrin repeat proteins (DARPins) on the viral capsid and carefully depleting DARPin-deficient particles, AAV vectors were made specific for Her2/neu, EpCAM or CD4. A single intravenous administration of vector targeted to the tumour antigen Her2/neu was sufficient to track 75% of all tumour sites and to extend survival longer than the cytostatic antibody Herceptin. CD4-targeted AAVs hit human CD4-positive cells present in spleen of a humanized mouse model, while CD8-positive cells as well as liver or other off-target organs remained unmodified. Mimicking conditions of circulating tumour cells, EpCAM-AAV detected single tumour cells in human blood opening the avenue for tumour stem cell tracking. Thus, the approach developed here delivers genes to target cell types of choice with antibody-like specificity.

Engineering the AAV capsid to optimize vector–host-interactions

“…Surprisingly, in sharp contrast to the negative influence of impaired HSPG binding on AAV2's infectivity in cell culture, these AAV2 capsids showed enhanced cardiac tropism when administered in vivo [24,26,27]. Interestingly, this unexpected novel in vivo tropism can be reversed by cell entry targeting as demonstrated recently [28 ]. Specifically, AAV2 particles carrying mutations in the HSPG binding motif (R585A, R588A) and additionally equipped with Design Ankyrin Repeat Proteins (DARPins) specific for Her2/neu or CD4 were able to precisely target human Her2/neu-positive tumors or human CD4-positive lymphocytes in vivo, without any detectable off-target activity [28 ].…”

“…Interestingly, this unexpected novel in vivo tropism can be reversed by cell entry targeting as demonstrated recently [28 ]. Specifically, AAV2 particles carrying mutations in the HSPG binding motif (R585A, R588A) and additionally equipped with Design Ankyrin Repeat Proteins (DARPins) specific for Her2/neu or CD4 were able to precisely target human Her2/neu-positive tumors or human CD4-positive lymphocytes in vivo, without any detectable off-target activity [28 ]. As illustrated by this example, in which the antibody-like specificity of DARPins had been employed to direct AAV2 vectors to a cell type of choice, rational design-based strategies are frequently explored in cell entry targeting.…”

An engineered serum albumin-binding AAV9 capsid achieves improved liver transduction after intravenous delivery in mice

“…Indeed, a large number of purified AAV9-ABDCon capsids were not observed to bind HSA on immuno-TEM. From our studies, it is unclear what fraction of capsids displayed the ABDCon peptide, although Münch and colleagues reported 68.6±10.2% recovery of His-tagged DARPIN-displaying AAV capsids using a HisTrap affinity chromatography column (28). We predict a similar chromatographic approach using HSA as the affinity ligand could be used to isolate and purify AAV9-ABDCon capsids.…”

Beyond Antibodies as Binding Partners: The Role of Antibody Mimetics in Bioanalysis

“…To enhance the efficiency of viral delivery systems, displaying DARPins on the surface of viral vectors was investigated by fusing DARPins with virus coat proteins and capsids (153–155). Münch et al (154) have developed a DARP infused adeno- associated viral delivery system without observing off-targeting effects due to the depletion of DARPin-deficient viral particles by using immobilized metal ion affinity chromatography. Their system has shown the capability of detecting tumors in vivo and circulating tumor cells in blood with high specificity and sensitivity.…”