Efficient transduction of primary vascular cells by the rare adenovirus serotype 49 vector

Neointima formation and vascular remodelling through vascular smooth muscle cell migration and proliferation can limit the long term success of coronary interventions, for example in coronary artery bypass grafting (CABG). Ex vivo gene therapy has the potential to reduce unnecessary cell proliferation and limit neointima formation in vascular pathologies. To date the species C adenovirus serotype 5 (Ad5) has been commonly used for pre-clinical gene therapy, however its suitability is potentially limited by relatively poor tropism for vascular cells and high levels of pre-existing immunity in the population. To avoid these limitations, novel species of adenovirus are being tested; here we investigate the potential of adenovirus 49 (Ad49) for use in gene therapy. Transduction of primary human vascular cells by a range of adenovirus serotypes was assessed; Ad49 demonstrated highest transduction of both vascular smooth muscle and endothelial cells. Gene transfer with Ad49 in vascular smooth muscle and endothelial cells was possible following short exposure times (*lt;1hr) and with low MOI which is clinically relevant. Ex vivo delivery to surplus CABG tissue showed efficient gene transfer with Ad49, consistent with the in vitro findings. Luminal infusion of Ad49GFP into intact CABG samples ex vivo resulted in efficient vessel transduction. In addition, no seroprevelance rates to Ad49 were observed in a Scottish cohort of patients from cardiovascular clinics, thus circumventing issues with pre-existing immunity. Our results show Ad49 has tropism for vascular cells in vitro and ex vivo and demonstrate Ad49 may be an improved vector for local vascular gene therapy compared to current alternatives

Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans) and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX), which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs). These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR) substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies), can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX), or alternatively, through the use of polymer-coated “stealth” vectors which avoid these interactions. Simultaneous retargeting and detargeting can be achieved by combining multiple genetic and/or chemical modifications

Evolution of magnetized, differentially rotating neutron stars: Simulations in full general relativity

We study the effects of magnetic fields on the evolution of differentially rotating neutron stars, which can form in stellar core collapse or binary neutron star coalescence. Magnetic braking and the magnetorotational instability (MRI) both redistribute angular momentum; the outcome of the evolution depends on the star's mass and spin. Simulations are carried out in axisymmetry using our recently developed codes which integrate the coupled Einstein-Maxwell-MHD equations. For initial data, we consider three categories of differentially rotating, equilibrium configurations, which we label normal, hypermassive and ultraspinning. Hypermassive stars have rest masses exceeding the mass limit for uniform rotation. Ultraspinning stars are not hypermassive, but have angular momentum exceeding the maximum for uniform rotation at the same rest mass. We show that a normal star will evolve to a uniformly rotating equilibrium configuration. An ultraspinning star evolves to an equilibrium state consisting of a nearly uniformly rotating central core, surrounded by a differentially rotating torus with constant angular velocity along magnetic field lines, so that differential rotation ceases to wind the magnetic field. In addition, the final state is stable against the MRI, although it has differential rotation. For a hypermassive neutron star, the MHD-driven angular momentum transport leads to catastrophic collapse of the core. The resulting rotating black hole is surrounded by a hot, massive, magnetized torus undergoing quasistationary accretion, and a magnetic field collimated along the spin axis--a promising candidate for the central engine of a short gamma-ray burst. (Abridged)Comment: 27 pages, 30 figure

Retargeting FX binding-ablated HAdV-5 to vascular cells by inclusion of the RGD-4C peptide in hexon hypervariable region 7 and the HI loop

Recent studies have generated interest in the function of human adenovirus serotype 5 (HAdV-5) hexon:  factor X (FX) binding and subsequent hepatocyte transduction and interaction with the immune system. Here, we retargeted adenovirus serotype 5 vectors, ablated for FX interaction, by replacing amino acids in hexon HVR7 with RGD-4C or inserting the peptide into the fibre HI loop. These genetic modifications in the capsid were compatible with virus assembly, and could efficiently retarget transduction of the vector via the αvβ3/5 integrin-mediated pathway, but did not alter immune recognition by pre-existing human neutralizing anti-HAdV-5 antibodies or by natural antibodies in mouse serum. Thus, FX-binding-ablated HAdV-5 can be retargeted but remain sensitive to immune-mediated attack. These findings further refine HAdV-5-based vectors for human gene therapy and inform future vector development

In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications

The human adenovirus phylogenetic tree is split across seven species (A–G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D—such as their cellular tropism, receptor usage, and in vivo biodistribution profile—remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)—a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications

Biodistribution and inflammatory profiles of novel penton and hexon double-mutant serotype 5 adenoviruses

The use of adenovirus serotype 5 (Ad5) vectors in the clinical setting is severely hampered by the profound liver tropism observed after intravascular delivery coupled with the pronounced inflammatory and innate immune response elicited by these vectors. Liver transduction by circulating Ad5 virions is mediated by a high-affinity interaction between the capsid hexon protein and blood coagulation factor X (FX), whilst penton-α(v)integrin interactions are thought to contribute to the induction of anti-Ad5 inflammatory and innate immune responses. To overcome these limitations, we sought to develop and characterise for the first time novel Ad5 vectors possessing mutations ablating both hexon:FX and penton:integrin interactions. As expected, intravascular administration of the FX binding-ablated Ad5HVR5*HVR7*E451Q vector (AdT*) resulted in significantly reduced liver transduction in vivo compared to Ad5. In macrophage-depleted mice, increased spleen uptake of AdT* was accompanied by an elevation in the levels of several inflammatory mediators. However ablation of the penton RGD motif in the AdT* vector background (AdT*RGE) resulted in a significant 5-fold reduction in spleen uptake and attenuated the antiviral inflammatory response. A reduction in spleen uptake and inflammatory activation was also observed in animals after intravascular administration of Ad5RGE compared to the parental Ad5 vector, with reduced co-localisation of the viral beta-galactosidase transgene with MAdCAM-1+ sinus-lining endothelial cells. Our detailed assessment of these novel adenoviruses indicates that penton base RGE mutation in combination with FX binding-ablation may be a viable strategy to attenuate the undesired liver uptake and pro-inflammatory responses to Ad5 vectors after intravascular deliver

Extension of Earth-Moon libration point orbits with solar sail propulsion

This paper presents families of libration point orbits in the Earth-Moon system that originate from complementing the classical circular restricted three-body problem with a solar sail. Through the use of a differential correction scheme in combination with a continuation on the solar sail induced acceleration, families of Lyapunov, halo, vertical Lyapunov, Earth-centred, and distant retrograde orbits are created. As the solar sail circular restricted three-body problem is non-autonomous, a constraint defined within the differential correction scheme ensures that all orbits are periodic with the Sun’s motion around the Earth-Moon system. The continuation method then starts from a classical libration point orbit with a suitable period and increases the solar sail acceleration magnitude to obtain families of orbits that are parametrised by this acceleration. Furthermore, different solar sail steering laws are considered (both in-plane and out-of-plane, and either fixed in the synodic frame or fixed with respect to the direction of sunlight), adding to the wealth of families of solar sail enabled libration point orbits presented. Finally, the linear stability properties of the generated orbits are investigated to assess the need for active orbital control. It is shown that the solar sail induced acceleration can have a positive effect on the stability of some orbit families, especially those at the L2 point, but that it most often (further) destabilises the orbit. Active control will therefore be needed to ensure long-term survivability of these orbits