Comparative analysis of lentiviral vectors and modular protein nanovectors for traumatic brain injury gene therapy

Abstract

Traumatic brain injury (TBI) remains as one of the leading causes of mortality and morbidity worldwide and there are no effective treatments currently available. Gene therapy applications have emerged as important alternatives for the treatment of diverse nervous system injuries. New strategies are evolving with the notion that each particular pathological condition may require a specific vector. Moreover, the lack of detailed comparative studies between different vectors under similar conditions hampers the selection of an ideal vector for a given pathological condition. The potential use of lentiviral vectors versus several modular protein-based nanovectors was compared using a controlled cortical impact model of TBI under the same gene therapy conditions. We show that variables such as protein/DNA ratio, incubation volume, and presence of serum or chloroquine in the transfection medium impact on both nanovector formation and transfection efficiency in vitro. While lentiviral vectors showed GFP protein 1 day after TBI and increased expression at 14 days, nanovectors showed stable and lower GFP transgene expression from 1 to 14 days. No toxicity after TBI by any of the vectors was observed as determined by resulting levels of IL-1β or using neurological sticky tape test. In fact, both vector types induced functional improvement per se.

Thy1‐YFP‐H Mice and the Parallel Rod Floor Test to Evaluate Short‐ and Long‐Term Progression of Traumatic Brain Injury

“…For instance, the adhesive tape test evaluates rat sensorimotor function after a CCI. This test is a very sensitive tool for the evaluation of neurological function beginning within a few hours and lasting up to 3 to 4 weeks after injury, but animals spontaneously recover thereafter and are not distinguishable from control animals (Chen, Hsu, Huang, & Wang, 2008;Negro-Demontel et al, 2014). A similar spontaneous functional recovery occurs within 2 to 3 weeks after TBI in mice when using both the rotarod test and the cylinder forelimb placement test (Clausen et al, 2011), or in rats using the rotarod test and beam walking test (Dixon et al, 1999;Yan, Hellewell, Bellander, Agyapomaa, & Morganti-Kossmann, 2011).…”

“…The CCI model of TBI is the most widely used model for the generation of defined, focal traumatic damage to the brain (d' Avila et al, 2012;Negro-Demontel et al, 2014;Osier & Dixon, 2016). It uses a computer-controlled piston mounted on a stereotaxic apparatus to induce a reproducible lesion.…”

“…A contusion depth of 4 mm induces a severe lesion that can be evaluated by different neurological tests. A smaller contusion depth can be used to evaluate histological or biochemical parameters but will induce less consistent neurological deficits as described previously (d'Avila et al, 2012;Negro-Demontel et al, 2014). Avoid extensive lesion of the hemisphere, as this does not represent most clinical cases.…”

Gene Delivery Strategies to Promote Spinal Cord Repair

“… 53 Alternatively, novel protein nanovectors have been developed and optimized for delivery to injured brain tissue. 172 Although not yet investigated in the spinal cord, protein-based carriers are attractive because they can be manipulated to incorporate biomolecules that target delivered genes to specific cell types or provide addition cues to promote tissue regeneration.…”

Integrated Method for Purification and Single-Particle Characterization of Lentiviral Vector Systems by Size Exclusion Chromatography and Tunable Resistive Pulse Sensing

“…For optimal quality control, single-particle analysis (SPA) would be preferred, since they allow following the distribution of key parameters (e.g., size) in the population, giving information about its heterogeneity [ 13 ]. Different approaches to SPA are in use (for comparison of methods see [ 13 , 14 ] and [ 15 ], among them electron microscopy (EM) and dynamic light scattering (DLS) approaches [ 16 ], nanoparticle tracking analysis (NTA) [ 11 , 14 , 17 , 18 ], flow virometry (FV) [ 19 , 20 ], and tunable resistive pulse sensing (TRPS) [ 13 , 21 , 22 ]. The latter is based on the Coulter principle, which states that particles pulled through a (physical) pore, while an electric current is applied, produce a change in impedance that is proportional to the volume of the particle traveling through the pore.…”