In vivo gene transfer with adenovirus vectors would significantly benefit from a tight control of the adenovirus-inherent liver tropism. For efficient hepatocyte transduction, adenovirus vectors need to evade from Kupffer cell scavenging while delivery to peripheral tissues or tumors could be improved if both scavenging by Kupffer cells and uptake by hepatocytes were blocked. Here, we provide evidence that a single point mutation in the hexon capsomere designed to enable defined chemical capsid modifications may permit both detargeting from and targeting to hepatocytes with evasion from Kupffer cell scavenging. Vector particles modified with small polyethylene glycol (PEG) moieties specifically on hexon exhibited decreased transduction of hepatocytes by shielding from blood coagulation factor binding. Vector particles modified with transferrin or, surprisingly, 5,000 Da PEG or dextran increased hepatocyte transduction up to 18-fold independent of the presence of Kupffer cells. We further show that our strategy can be used to target high-capacity adenovirus vectors to hepatocytes emphasizing the potential for therapeutic liver-directed gene transfer. Our approach may lead to a detailed understanding of the interactions between adenovirus vectors and Kupffer cells, one of the most important barriers for adenovirus-mediated gene delivery.
Capsid surface shielding of adenovirus vectors with synthetic polymers is an emerging technology to reduce unwanted interactions of the vector particles with cellular and non-cellular host components. While it has been shown that attachment of shielding polymers allows prevention of undesired interactions, it has become evident that a shield which is covalently attached to the vector surface can negatively affect gene transfer efficiency. Reasons are not only a limited receptor-binding ability of the shielded vectors but also a disturbance of intracellular trafficking processes, the latter depending on the interaction of the vector surface with the cellular transport machinery. A solution might be the development of bioresponsive shields that are stably maintained outside the host cell but released upon cell entry to allow for efficient gene delivery to the nucleus. Here we provide a systematic comparison of irreversible versus bioresponsive shields based on synthetic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. In addition, the chemical strategy used for generation of the shield allowed for a traceless bioresponsive shielding, i.e., polymers could be released from the vector particles without leaving residual linker residues. Our data demonstrated that only a bioresponsive shield maintained the high gene transfer efficiency of adenovirus vectors both in vitro and in vivo. As an example for bioresponsive HPMA copolymer release, we analyzed the in vivo gene transfer in the liver. We demonstrated that both the copolymer's charge and the mode of shielding (irreversible versus traceless bioresponsive) profoundly affected liver gene transfer and that traceless bioresponsive shielding with positively charged HPMA copolymers mediated FX independent transduction of hepatocytes. In addition, we demonstrated that shielding with HPMA copolymers can mediate a prolonged blood circulation of vector particles in mice. Our results have significant implications for the future design of polymer-shielded Ad and provide a deeper insight into the interaction of shielded adenovirus vector particles with the host after systemic delivery.
Adenoviruses (Ad) are promising vectors for therapeutic interventions in humans. Despite significant knowledge regarding the biology of Ad interactions with cells in vitro, the molecular mechanisms governing in vivo Ad infectivity and bio-distribution remain poorly understood. Pharmacokinetic studies of Ad vectors after intravascular delivery demonstrate that the majority of an administered virus dose is rapidly sequestered from the circulation by the liver. Earlier we showed that Ad particles are distributed in liver tissue amongst three distinct cellular compartments, namely i) parenchymal liver cells-hepatocytes, ii) hepatic residential macrophages, Kupffer cells, and iii) hepatic sinusoid endothelial cells. Importantly, we found that the ablation of Ad interaction with only one of these cellular compartments cannot prevent virus trapping the liver shortly after intravascular virus injection and, likely, results in compensatory redistribution of the virus among two remaining cellular compartments, thus functionally ensuring the quantitative removal of the virus from the blood. We speculated that to prevent Ad sequestration by the liver after intravascular administration, simultaneous ablation of virus interaction with all three cellular compartments of the liver is ultimately required. To evaluate this assumption experimentally, we introduced in a single human Ad5based vector specific mutations that abrogate virus interactions with one, two, or all three hepatocellular compartments. To ablate Ad interaction with hepatocytes in vivo, we introduced T425A mutation in hexon hyper-variable loop HVR7 that completely abrogates virus interaction with blood coagulation FX. To prevent Ad interaction with liver sinusoid endothelial cells, we introduced a substitution of the RGD-motif-containing penton loop for an iso-functional integrininteracting non-RGD-containing peptide. We also serendipitously found that the deletion of hexon HVR1 region, in addition to penton RGD-loop substitution and T425A HVR7 mutation, results in the reduction of virus interaction with Kupffer cells. Intravenous injection into mice of Ad variants containing individual mutations either in the penton or hexon or a vector with only two mutations in a single vector does not prevent sequestration of resultant Ad in the liver. However, Ad variant, containing all three mutations simultaneously, completely escaped being sequestered in the liver after intravascular injection and, instead, 20-fold higher amounts of the virus were recovered from the spleen, indicating the major change in virus bio-distribution, compared to unmodified vectors with a wild-type capsid, or vectors with individual mutations in either penton or hexon. To our knowledge this is the first demonstration of a feasibility of constructing Ad variants that would escape liver sequestration after intravascular administration. These variants may prove to be a useful platform for gene delivery to extra-hepatic cells and/or targeting disseminated metastatic cancers.
Kit cITD is a complex internal tandem duplication involving the juxtamembrane (JMD) and kinase domain of stem cell factor receptor c-kit which was identified recently in 7% of childhood AML. kit cITD induces constitutive proliferation and apoptosis resistance that can be synergistically blocked with imatinib (im) and rapamycin (ra). Due to a long half-life ra has disadvantageous pharmacological characteristics making drug handling especially difficult in children with serum concentrations quickly raising to toxic levels. We therefore compared ra with its ester analogue everolimus (RAD001, ev) which has a better bioavailability and a shorter half-life in order to assess equivalence and potential functional superiority. Ba/F3 cells, stably transfected with c-kit WT and kit cITD, were treated with im, ra and ev alone and in combination. In proliferation assays of WT and cITD cell lines ev was a significantly more potent inhibitor than ra. When ra or ev were combined with low im concentrations proliferation was synergistically inhibited with both drugs but again significantly better with ev. In cITD expressing cells ra and ev alone were comparably poor inducers of apoptosis. In assays combining im with ra or ev both combinations showed a significant and comparable synergism with an additional dose effect of the mTOR inhibitors between 3nM and 0.3 nM. In a NOD/SCID animal model all animals injected with kit cITD succumbed due to multi-organ infiltration of c-kit positive cells within 20 days. Survival was prolonged in animals treated with im/ra and ev. We therefore conclude that ev is at least equivalent to ra and can replace ra exploring a molecular based therapeutic attempt in susceptible children with identified mutations of the JMD of c-kit.
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