Age and lesion-induced increases of GDNF transgene expression in brain following intracerebral injections of DNA nanoparticles

Abstract

In previous studies that used compacted DNA nanoparticles (DNP) to transfect cells in the brain, we observed higher transgene expression in the denervated striatum when compared to transgene expression in the intact striatum. We also observed that long term transgene expression occurred in astrocytes as well as neurons. Based on these findings, we hypothesized that the higher transgene expression observed in the denervated striatum may be a function of increased gliosis. Several aging studies have also reported an increase of gliosis as a function of normal aging. In this study we used DNPs that encoded for human glial cell line-derived neurotrophic factor (hGDNF) and either a non-specific human polyubiquitin C (UbC) or an astrocyte-specific human glial fibrillary acidic protein (GFAP) promoter. The DNPs were injected intracerebrally into the denervated or intact striatum of young, middle-aged or aged rats, and GDNF transgene expression was subsequently quantified in brain tissue samples. The results of our studies confirmed our earlier finding that transgene expression was higher in the denervated striatum when compared to intact striatum for DNPs incorporating either promoter. In addition, we observed significantly higher transgene expression in the denervated striatum of old rats when compared to young rats following injections of both types of DNPs. Stereological analysis of GFAP+ cells in the striatum confirmed an increase of GFAP+ cells in the denervated striatum when compared to the intact striatum and also an age-related increase; importantly, increases in GFAP+ cells closely matched the increases in GDNF transgene levels. Thus neurodegeneration and aging may lay a foundation that is actually beneficial for this particular type of gene therapy while other gene therapy techniques that target neurons are actually targeting cells that are decreasing as the disease progresses.

Impact of age and vector construct on striatal and nigral transgene expression

“…We used a novel combination of RNAscope in situ hybridization for the viral CβA promoter 35 and immunohistochemistry for astrocytes (S100 calcium-binding protein B, S100B) or neurons (Neuronal Nuclei, Neu-N) to visualize the cellular location of viral particles in the transduced zone in the striatum. Despite previous evidence suggesting an increase in the population of astrocytes in the striatum with aging, 36 no qualitative increase in S100B astrocytes was observed in the aged compared to the young striatum. Further, in both the young adult and aged brain, for all vector constructs, viral genomes overwhelmingly colocalized with NeuN-immunoreactive neurons (Figure 5a–c and Figure 5g–i), indicating their presence within neurons, corresponding with our GFP-expression data (Supplementary Figures S4 and S6).…”

“…Previous studies have reported a doubling of astrocytes in the striatum of aged as compared to young adult rats. 36 To investigate the possibility that reduced transgene expression in the aged striatum is due to increased numbers of astrocytes sequestering viral genomes from neurons, we analyzed the number of viral particles within neurons vs. astrocytes. We did not observe any appreciable qualitative differences in the number of astrocytes in the young adult versus aged rat striatum.…”

Destination Brain: the Past, Present, and Future of Therapeutic Gene Delivery

“…Complexing NP with polyethylene glycol (PEG), or PEGylation, increased transfection efficiencies, reduced toxicity, and imparted “stealth” properties (Xin et al, 2012; Rungta et al, 2013; Morris and Labhasetwar, 2015; Yurek et al, 2015). Convection-enhanced delivery also increased efficiency of NP administration (Mastorakos et al, 2015).…”

“…Neurons and glia were transfected and sustained expression for over 11 weeks (Yurek et al, 2009). Transfection efficiency was higher in denervated striatum of 6-OHDA-induced mice and old mice, highlighting their disease sensitivity and age as a factor in gene delivery (Yurek et al, 2015). A cyclized-knot polymer delivered GDNF to primary rat astrocytes and the astrocyte cell line Neu7 that led to functional effects in co-cultured dorsal root ganglion cells (Newland et al, 2013).…”