Improvement of HSV-1 based amplicon vectors for a safe and long-lasting gene therapy in non-replicating cells

Abstract

A key factor for developing gene therapy strategies for neurological disorders is the availability of suitable vectors. Currently, the most advanced are adeno-associated vectors that, while being safe and ensuring long-lasting transgene expression, have a very limited cargo capacity. In contrast, herpes simplex virus-based amplicon vectors can host huge amounts of foreign DNA, but concerns exist about their safety and ability to express transgenes long-term. We aimed at modulating and prolonging amplicon-induced transgene expression kinetics in vivo using different promoters and preventing transgene silencing. To pursue the latter, we deleted bacterial DNA sequences derived from vector construction and shielded the transgene cassette using AT-rich and insulator-like sequences (SAm technology). We employed luciferase and GFP as reporter genes. To determine transgene expression kinetics, we injected vectors in the hippocampus of mice that were longitudinally scanned for bioluminescence for 6 months. To evaluate safety, we analyzed multiple markers of damage and performed patch clamp electrophysiology experiments. All vectors proved safe, and we managed to modulate the duration of transgene expression, up to obtaining a stable, long-lasting expression using the SAm technology. Therefore, these amplicon vectors represent a flexible, efficient, and safe tool for gene delivery in the brain.

An HSV-1-H129 amplicon tracer system for rapid and efficient monosynaptic anterograde neural circuit tracing

“…Anterograde monosynaptic tracing occurs only when the tracer (represented by H129 Amp -CTG) and the helper co-infect the same neurons when are simultaneously administrated into the brain area of interest. These viral tracer components do not distinguish between outputs of different neuron types in the brain area with the optimized dosage and ratio of the tracer/helper co-infected starter neurons 19 , 29 (Fig. 1e ).…”

“…Infection of HSV amplicon may initiate a cascade of immune responses including expression of Type I IFNs, which have been reported to suppress transgene expression at the transcriptional level by activation of STAT1 41 , 42 . In addition, the presence of prokaryotic DNA sequences in the pseudo-genome of H129 Amp tracer may also lead to transcriptional silencing of the entire vector sequence due to the inactive form of chromatin immediately after infection 19 , 29 . The transgene expression kinetics of HSV amplicon has been shown to peak quickly (1 day post-injection, dpi) and then drop by over 3 logs within a week 19 .…”

“…In addition, the presence of prokaryotic DNA sequences in the pseudo-genome of H129 Amp tracer may also lead to transcriptional silencing of the entire vector sequence due to the inactive form of chromatin immediately after infection 19 , 29 . The transgene expression kinetics of HSV amplicon has been shown to peak quickly (1 day post-injection, dpi) and then drop by over 3 logs within a week 19 . We observe fluorescence labeling of the H129 Amp tracer system as early as 1 day post-injection at the injection site, but this signal quickly dims below the detection threshold for confocal imaging at 7 days post-injection.…”

“…Other improvements could be made by adding insulator and AT-rich sequences or by removing the bacterial sequence in the amplicon plasmid. These modifications could extend the labeling duration 19 . Second, toxicity does occur in the starter neurons that are co-infected by H129 Amp tracer and helper.…”

“…Ori plays a critical role in initiating the amplicon plasmid replication and forming the concatameric pseudo-genome, and pac is essential for the pseudo-genome recognition and packaging into the viral capsid 17 , 18 . The HSV-1 amplicon shows promise as a gene transfer vector based on its unique features: (i) broad cell tropism that mimics the infectivity of wild-type HSV-1; (ii) large transgene capacity that supports a maximum of ~150 kb genetic payload 19 , 20 ; (iii) high transgene expression efficiency, expressing the target gene(s) simultaneously with the multiunit expression cassettes (multiple-copy of target); and (iv) vastly minimized toxicity as it neither replicates nor produces toxic viral proteins 16 , 20 .…”