Better Targeting, Better Efficiency for Wide-Scale Neuronal Transduction with the Synapsin Promoter and AAV-PHP.B

Abstract

Widespread genetic modification of cells in the central nervous system (CNS) with a viral vector has become possible and increasingly more efficient. We previously applied an AAV9 vector with the cytomegalovirus/chicken beta-actin (CBA) hybrid promoter and achieved wide-scale CNS transduction in neonatal and adult rats. However, this method transduces a variety of tissues in addition to the CNS. Thus we studied intravenous AAV9 gene transfer with a synapsin promoter to better target the neurons. We noted in systematic comparisons that the synapsin promoter drives lower level expression than does the CBA promoter. The engineered adeno-associated virus (AAV)-PHP.B serotype was compared with AAV9, and AAV-PHP.B did enhance the efficiency of expression. Combining the synapsin promoter with AAV-PHP.B could therefore be advantageous in terms of combining two refinements of targeting and efficiency. Wide-scale expression was used to model a disease with widespread pathology. Vectors encoding the amyotrophic lateral sclerosis (ALS)-related protein transactive response DNA-binding protein, 43 kDa (TDP-43) with the synapsin promoter and AAV-PHP.B were used for efficient CNS-targeted TDP-43 expression. Intracerebroventricular injections were also explored to limit TDP-43 expression to the CNS. The neuron-selective promoter and the AAV-PHP.B enhanced gene transfer and ALS disease modeling in adult rats.

Distinct in vivo roles of secreted APP ectodomain variants APP sα and APP sβ in regulation of spine density, synaptic plasticity, and cognition

“…In the CA3 region, APPsa expression appeared slightly lower compared to the expression obtained in pyramidal cells of the subiculum, the CA1 ( Fig 1C) and the CA2 regions. Consistent with previous studies (Jackson et al, 2016), we noted that APPsa was detectable in HEK cells transfected with synapsin promoter driven AAV constructs (see Fig EV2A), likely due to AAV plasmid overload. In vivo, however, AAV-APPsa expression was restricted to neurons, as shown by double immunofluorescence staining against the HA-tag and the neuronal marker NeuN (Fig 1D and E).…”

Small Alphaherpesvirus Latency-Associated Promoters Drive Efficient and Long-Term Transgene Expression in the Central Nervous System

“…For example, CNS therapies for Pompe disease 56 and Parkinson's disease 57 are based on delivery of relatively large genes such as GAA (2.9 Kb) and GDNF (2.5 Kb). For these and other similar cases, the use of small, non-repressible promoters are ideal replacements for larger promoters or even smaller CMV and hSyn promoters shown to be quickly repressed after delivery 14,15 .…”

“…Accordingly, the discovery of short promoter sequences that sustain strong and long-lived transcription is paramount to expand the transgene payload and achieve chronic therapeutic effect with one viral dose.Several strong promoters such as neuron-specific enolase (NSE, 1800 bp) 10, 11 , calcium/calmodulin-dependent protein kinase II alpha (CaMKIIa, 1300 bp) 12 and human elongation factor 1 alpha (EF1α, 1264 bp) 12,13 have been used in systemic AAV delivery 6 .However, the considerable size of these promoter sequences limits the use of large therapeutic transgenes or multiple small transgenes. Moreover, short promoters such as the human cytomegalovirus immediate-early enhancer and promoter (CMV, 600 bp) 14 or truncated versions of the human synapsin promoter (hSyn, 468 bp) 15 , are considerably weaker to drive gene transcription and expression, and in some cases, are completely repressed or inactivated only weeks after delivery 13,[15][16][17][18] . Similarly, small ubiquitous promoters like beta glucuronidase (GUSB, 378 bp) 19 or ubiquitin C (UBC, 403 bp) 13, 20 have shown weak transcription levels.Here, we describe and validate three alphaherpesvirus latency-associated promoters (LAP), called LAP1 (498 bp), LAP2 (404 bp) and LAP 1_2 (880 bp) obtained from the genome of the herpesvirus pseudorabies virus (PRV).…”

“…However, the considerable size of these promoter sequences limits the use of large therapeutic transgenes or multiple small transgenes. Moreover, short promoters such as the human cytomegalovirus immediate-early enhancer and promoter (CMV, 600 bp) 14 or truncated versions of the human synapsin promoter (hSyn, 468 bp) 15 , are considerably weaker to drive gene transcription and expression, and in some cases, are completely repressed or inactivated only weeks after delivery 13,[15][16][17][18] . Similarly, small ubiquitous promoters like beta glucuronidase (GUSB, 378 bp) 19 or ubiquitin C (UBC, 403 bp) 13, 20 have shown weak transcription levels.…”

Adeno‐associated virus‐based Alzheimer's disease mouse models and potential new therapeutic avenues

“…The improved neuronal targeting by AAV.PHP.B was observed in the peripheral nervous system and was leveraged for gene therapy in a mouse model of synucleinopathy (Morabito et al, ). AAV.PHP.B delivery was moderately more or similarly efficient as compared to its parent AAV9 in adult rats (Jackson, Dayton, Deverman, & Klein, ) or NHP (Hordeaux et al, ; Matsuzaki et al, ). A dose response study in adult rats by i.v.…”