Application of rAAVs ex vivo and in vivo

Ex vivo application of rAAV serotypes to elucidate their potential for gene therapy

Human mesenchymal stem cells - Mesenchymal stem cells (MSCs) have attracted atten-tion as a vehicle for gene therapy in regenerative medicine with their multi-potent ability to differentiate into various cell types, e.g., adipocytes and neurons (Mohammadian et al., 2016).

Analogous to our experiment in which we gained the expression profile of our five rAAV serotypes on immortalized human cell lines, we characterised them on MSCs derived from the Warton´s jelly of the umbilical cord of three donors. Interesting to our knowledge was

the diversity of susceptibility not only between the serotypes but also between the donors.

rAAV serotype 2 robustly performed on all three donor MSCs what might explain the preferred usage of this serotype in recent studies (Stender et al., 2007). Nevertheless, the serotype 6 and the chimeric 1/2 partially achieved a higher transduction rate. According to our data presented here, we strongly suggest investigators to elucidate patient-wise which serotype is superior.

As a follow-up to this study we planned to elucidate whether the gene manipulation pre-differentiation to neurons and osteoblasts keeps constant or is silenced.

Cortical cultures of the Wistar rat and three non-human primates - With the ethical obligation to minimize the need of in vivo experiments and animals as tissue donors for ex vivo experiments we tested our rAAVs on cortical cultures of the Wistar rat and three non-human primates in the next step for the neuronal delivery. The tissue came from animals which had been euthanised for other reasons and where the cortex was not required for further examinations. This, especially for the non-human primates, limited the excess to the material, especially from young individuals.

Acute cortex slices could be cultivated from all species, whereas dissociated cultures could only be gained from the Wistar rat. As tissue from younger individuals has a more extended viability in culture, we assume that the material from even newborn or adult non-human primates is not suitable to lay out a culture. The expression profile of the five serotypes with constitutive and neuron-specific promotors on dissociated cultures pointed out that the serotype 5 mainly infects glia cells (based on morphological cell type identification). It surprisingly also drives an expression with the CaMKII promotor that is widely claimed as neuron-specific (Taymans et al., 2007). The rAAV2 was the only serotype transducing no cells.

These results are in agreement with Howard et al. (2008) who also found no neuron-related expression using another constitutive, the CMV promotor for rAAV5. They described a late and rare onset of expression for the serotype 2, which might have been out of our observation period.

Expression was only detected in few cases when an opsin was included besides the reporter gene. Our study illustrates that, e.g., serotype 1 and the chimeric 1/2 are broadly effective ex vivo and can also be used in voltage clamp experiments. But we also reasoned that rAAV5 with constructs for neuronal targeting needs to be applied in vivo or, e.g., on cerebral organoid models of the brain (Lancaster et al., 2013).

In vivo applications

Transduction of rAAV5 in the mouse cochlea - The cochlea is a well-established target for rAAVs as for the aim to restore hearing with optogenetic implants (György et al., 2017; Moser, 2015). In support of the above-discussed results this offered us the oppor-tunity to assess the in vivo potential of our viral batches. We injected the two high titer viruses that were also addressed in the quality control section (see 4.3) in the cochlea of mice and visualised the transduced cells in immunofluorescence stainings. Both viruses transduced some supporting cells. No high number of transduced cells was expected thus the serotype 5 is none of the preferred serotypes for cochlea transduction. For the CAG promotor, it is published that it can drive a high expression in murine cochlea cells (Liu et al., 2007). Based on the micrographs we did not identify any changes at the injection sites and thereby conclude that the viral samples are safe for application.

Transduction of rAAV5 in the cortex of Macaca mulatta -In the course of an experiment in our cooperation with the Cognitive Neuroscience Laboratory and the Neurobiology Laboratory (Deutsches Primatenzentrum GmbH, Göttingen), which focused on the injec-tion strategy and viral spread, a purchased and a self-produced virus were injected in the dorsal and ventral pre-motor cortex of aMacaca mulatta. The animal was euthanised ten weeks post-virus injection and the brain regions processed for immunofluorescence stain-ings. Based on the detected transgene expression of the reference virus, we concluded that the injection procedure was working. For the self-produced virus, no transgene expression related to this virus was visualised in the adjacent neuropil but alterations regarding a lower background fluorescence intensity and a reduction of NeuN positive cells. We can thereby make no conclusions about the virus functionality in the cortex of Macaca mu-latta.

The alterations of the neuropil can have various reasons: a mechanic injury caused by the injection, neuropathogen virus infection, ischemia, anoxia or hypoglycemia (Vandevelde et al., 2012). For a detailed investigation of the trigger, advanced histological stainings could be performed to examine, e.g., the glia cells‘ reaction. Additionally, control in-jections of the virus sample buffer alone and a control substance, like PBS, could be revealing.