Ilia Fishbein, MD, PhD

Description of Research Expertise

In general terms my main research interests are in the field of vascular gene therapy.

My postdoctoral studies explored a strategy of local arterial wall delivery of adenoviral vectors immobilized on the stent surface. Using monomolecular layer of metal-coordinating polymer as a bridging moiety we were able to achieve covalent attachment of adenoviral affinity adapters (either anti-adenoviral antibody, or D1 domain of the native Cocxackie-Adenovirus Receptor; CAR) to metal surfaces. Subsequent reversible vector tethering allows for highly efficient spatially restricted gene transfer in vitro and has shown promising results in rat and pig angioplasty/stenting model, demonstrating significant reporter gene activity in all layers of stented arterial segment.

Lately we investigated an alternative strategy of direct covalent vector tethering to the metal surface via hydrolysable cross-linker, which might be advantageous since it seems to be more robust and versatile in terms of vector type. Presently we are conducting large animal study with therapeutic adenoviral vector (Ad-eNOS) to assess antirestenotic efficacy of gene delivery stents in the pig angioplasty/stenting model.

Additionally we have recently developed a novel strategy for localized delivery of viral and non-viral gene therapy vectors co-formulated with magnetic nanoparticles to stented arteries using uniform magnetic field-induced targeting. The latter approach allows as well a targeted delivery of cells transduced with the abovementioned vectors to stented arteries, facilitating the process of reendothelialization.

Gene delivery from biodegradable stents
Recent advent of drug-eluting metal stents has significantly improved short-term and intermediate outcomes in patients undergoing coronary stenting. However, animal and clinical data shows that long-term efficacy of this new treatment is questionable. Most experts in the field agree that a presence of a stent per se perpetuate chronic foreign body response leading to eventual revascularization failure. To this end, attempts have been made to formulate stent fabricated from biodegradable polymers. Polyesters (e.g. polylactic acid; PLA) possess mechanical properties comparable to metals and thus represent uppermost interest. Fabrication of PLA stents is compatible with either bulk immobilization, or surface tethering of adenoviral vectors via cleavable cross-linker developed in our lab. Therapeutic gene constructs delivered from degradable stent platform might address excessive smooth muscle proliferation, migration and synthetic activity or/and accelerate stent decomposition.

Vascular homing of genetically modified macrophages
Cells of monocyte/macrophage lineage play central role in the initiation of atherosclerotic lesions due to selective homing in the areas of dysfunctional endothelium and vascular inflammation. Likewise, macrophages accumulate in the injured/stented arteries and induce smooth muscle cell activation owing to the secretion of PDGF, bFGF and several other growth factors and cytokines. Strategies directed on temporary elimination or functional impairment of macrophages have been shown to reduce restenosis in animal models.

I would like to exploit intrinsic vasculature homing traits of monocytes to achieve selective cell/gene delivery to evolving atherosclerotic and restenotic lesions. The suggested approach is based on stable genetic modification of autologous monocytes ex vivo with subsequent systemic administration in hypercholesterolemic transgenic Apo E-deficient mice and in rats with injury-induced restenosis. A list of possible genetic modification of monocytes comprises the loss of function interventions inhibiting the synthesis/secretion of growth factors or downregulation of NFkB and TNFa to block inflammatory cascade, or the gain of function gene therapy emphasizing secreted antiproliferative gene products.

Surface modification of adenoviral vectors
Cell types constituting vasculature has relatively low susceptibility to adenoviral infection, primarily because of limited CAR expression. Moreover, duration of transgene expression in the arterial wall does not usually exceed several weeks due to marked immune response elicited by adenoviruses. To address these two problems I recently examined a strategy of adenoviral vector surface modification by polyallylamine-based polymer derivatized with photo-reactive (benzophenone) and thiol-reactive (pyridyl-dithio) groups. Upon 30 sec UV irradiation of the virus/polymer mixture activated benzophenone groups covalently tether polymer to the capsid proteins of virus vector. Thiol-reactive moieties of the virus-conjugated polymer were further derivatized with protein transduction domain peptides, TAT and Antp. The latter were shown to mediate facilitated cellular internalization of liposomes and viruses notwithstanding of specific receptor repertoire. Our first in vitro experiments with TAT/Antp-modified adenoviral vectors have shown more than 100-fold increase in reporter gene transduction in comparison with non-modified vector in the cell line of endothelial origin.

We also predict enhanced in vivo activity of modified vector since surface-bound polymer fibers should create protective layer hindering virus inactivation by neutralizing antibodies, as was previously shown for polyethylene glycol-modified adenovirus.

Lastly, the suggested strategy will allow us to co-modify vector surface with other relevant targeting ligands thus increasing tissue residence of the adenovector and its specificity for the activated endothelium and smooth muscle cells.