Spinal Cord Molecular and Cellular Changes Induced by Adenoviral Vector- and Cell-Mediated Triple Gene Therapy after Severe Contusion

Abstract

The gene therapy has been successful in treatment of spinal cord injury (SCI) in several animal models, although it still remains unavailable for clinical practice. Surprisingly, regardless the fact that multiple reports showed motor recovery with gene therapy, little is known about molecular and cellular changes in the post-traumatic spinal cord following viral vector- or cell-mediated gene therapy. In this study we evaluated the therapeutic efficacy and changes in spinal cord after treatment with the genes encoding vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), angiogenin (ANG), and neuronal cell adhesion molecule (NCAM) applied using both approaches. Therapeutic genes were used for viral vector- and cell-mediated gene therapy in two combinations: (1) VEGF+GDNF+NCAM and (2) VEGF+ANG+NCAM. For direct gene therapy adenoviral vectors based on serotype 5 (Ad5) were injected intrathecally and for cell-mediated gene delivery human umbilical cord blood mononuclear cells (UCB-MC) were simultaneously transduced with three Ad5 vectors and injected intrathecally 4 h after the SCI. The efficacy of both treatments was confirmed by improvement in behavioral (BBB) test. Molecular and cellular changes following post-traumatic recovery were evaluated with immunofluorescent staining using antibodies against the functional markers of motorneurons (Hsp27, synaptophysin, PSD95), astrocytes (GFAP, vimentin), oligodendrocytes (Olig2, NG2, Cx47) and microglial cells (Iba1). Our results suggest that both approaches with intrathecal delivery of therapeutic genes may support functional recovery of post-traumatic spinal cord via lowering the stress (down regulation of Hsp25) and enhancing the synaptic plasticity (up regulation of PSD95 and synaptophysin), supporting oligodendrocyte proliferation (up regulation of NG2) and myelination (up regulation of Olig2 and Cx47), modulating astrogliosis by reducing number of astrocytes (down regulation of GFAP and vimetin) and microglial cells (down regulation of Iba1).

Triple-Gene Therapy for Stroke: A Proof-of-Concept in Vivo Study in Rats

“…Immunofluorescent analysis revealed expression of VEGF, GDNF, and NCAM in neural and glial cells at the site of stroke ( Figure 6 ). Notably, the data on the level of the reporter and therapeutic genes expression correspond to the short period of expression for adenoviral vectors and are consistent with previously described results ( Izmailov et al, 2017 ).…”

“…UCB-MC have their own endogenous potential to increase survivability of the affected neural and glial cells in the penumbra. The results are consistent with our previous data demonstrating the positive effect of UCB-MC on regeneration of spinal cord after severe contusion ( Izmailov et al, 2017 ). Based on these results and on the efficacy of direct triple gene therapy for stroke we proposed to enhance the therapeutic potential of UCB-MC via simultaneous transduction of UCB-MC with Ad-VEGF, Ad-GDNF, and Ad-NCAM.…”

“…To increase the targeting of UCB-MC into the nervous tissue we used Ad5-NCAM where NCAM is thought to be responsible for targeting, migration, and viability of the transplanted UCB-MC. In our previous research we documented this targeting of UCB-MC in lumbar spinal cord after intravenous ( Safiullov et al, 2015 ) and intrathecal injection ( Izmailov et al, 2017 ). Meanwhile, localization of the gene modified UCB-MC does not diminish their therapeutic efficacy since the biologically active recombinant molecules can be easily circulated by CSF to affected areas.…”

“…As expected, cerebrospinal fluid (CSF) flow facilitated dissemination of the viral vectors and UCB-MC+Ad-GFP throughout the CNS. In a previous study, we demonstrated expression of reporter GFP gene along the spinal cord following intrathecal injection of Ad-GFP ( Izmailov et al, 2017 ). In the present study, the specific fluorescence of GFP in brain cells was visualized at the site of stroke and in the opposite hemisphere after viral-mediated GFP gene delivery ( Figure 5A ).…”

Newly regenerated axons through a cell-containing biomaterial scaffold promote reorganization of spinal circuitry and restoration of motor functions with epidural electrical stimulation

“…Current therapies for spinal cord injury (SCI) demonstrate only limited functional recovery. Recent advances in bioelectronics and tissue engineering make available novel neuromodulatory and neuroregenerative therapies, aimed to improve function after SCI [1][2][3][4][5] . Encouraging results using epidural electrical stimulation (EES) to restore motor function in humans with SCI [6][7][8] have been attributed to the presence of functionally silent fibers and the combination of EES and motor training [9][10][11] .…”