rAAV2 pep1jhf demonstrates efficacy in CAM assays

5. Results and Discussion

5.2. Peptides binding the EGFR dimerization arm

5.2.10. rAAV2 pep1jhf demonstrates efficacy in CAM assays

Another focus lies on the analysis of rAAV with integrated peptide sequences. Larger viral prepa-rations were produced and CAM assays were performed by our collaboration partners. For rAAV2 pep1osy no adverse effects have been observed after systemic injection. In comparison the injection of rAAV2 pep1jhf led to a high mortality of the embryos (7 out of 15) and surviving animals showed severe cerebral haemorrhages and gastric bleeding. The blood clotting time in chicken em-bryos was shown to be slow and thus, also slight bleedings can be lethal.¹²⁴ As two different AAV preparations and furthermore a reduced vector amount (5×10¹⁰ vectors per egg) were used for these experiments, we can exclude that contaminants or a too high dose led to this severe phenotype.

Rational design of peptides was performed using the PDB model 1IVO which represents the crystal structure of the human EGFR. The chicken EGFR shares about 70% sequence homology with the human EGFR. Taking a closer look into the interaction between the designed peptides and the crystal structure of the human EGFR demonstrates the binding interface with atomic contacts based

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39 on the van der Waals (VDW) radii (Figure 26A, B). Comparing the regions of contacts with the sequence alignment of human and chicken EGFR indicates that interacting regions are almost iden-tical between the organisms (Figure 26C). Thus, transduction not only of the human EGFR in the tumor tissue of A431 cells should be possible, also the transduction of chicken EGFR in the whole organism might be feasible.

Figure 26: Identification of the binding interface of pep1jhf and pep1osy. PDB files of the bound peptide to the EGFR are the results of the rational peptide design of pep1jhf (A) and pep1osy (B). Contacts between atom pairs of peptide and receptor were determined using UCSF Chimera. A cut-off of 1.0 Å distance was defined to identify atoms pairs.

The binding interface is highlighted in magenta (peptide) and dark blue (EGFR). (C) Alignment of the human EGFR (UniProt P00533) and the chicken EGFR (UniProt A0A1D5NZB4) was generated using Geneious. Regions identified from previous contact determination are highlighted in magenta.

Figure 27A-F represents the results of qPCR analysis for detection of delivered genomic copies to the respective organs. Again systemic PBS injection served as negative control and here transduc-tion was not detected. For rAAV2 pep1osy almost no transductransduc-tion was observed in any tissue. This was not expected as previous cell culture experiments demonstrated that receptor binding, intracel-lular trafficking, nuclear entry and genome conversion is possible upon integration of the targeting peptide. The most important difference between the previous cell culture experiments and the in vivo model lies within the blood clearance and the vascular escape. Since, peptides should be able to interact not only with the human EGFR, but also with the chicken EGFR, rAAV2 variants should also be detected in EGFR-expressing tissues The inefficient transduction of tissues by rAAV2 pep1osy can thus only be explained via the blood clearance and the vascular escape.

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pep1jhf pep1osy

The qPCR analysis for rAAV2 pep1jhf showed genomic copies in all organs of the chicken embryo even elevated compared to transduction with the wild-type rAAV2 capsid. The EGFR is widely overexpressed in foetal tissues and thus transduction of all organs is likely. The high lethality after systemic injection due to severe cerebral haemorrhages and gastric bleeding might be explained by qPCR results. The highest amounts of genomic copies were found in these organs. The high amount in the brain is surprising as for the rAAV2 wild-type variant no transduction was observed and it is known that rAAV2 is not able to cross the blood-brain barrier.¹²³ However, it was shown that the blood-brain barrier of one-day old chicken is humiliated, which might allow for transcytosis of the re-targeted rAAV vector.¹²⁵ EGFR expression in the foetal brain is elevated which might contribute to the high transduction efficiency. Gastric bleeding on the other hand was also rarely observed in patients for other EGFR-targeting pharmaceuticals already in clinic.^126,127 Bleeding in the embryo might be the result from an effective EGF receptor blocking. Interruption of the downstream sig-naling is involved in different cellular processes and if the receptor is not only overexpressed in the tumor but also in the embryo’s organs this can have severe side-effects.

Figure 27: Determination of viral genomes for rAAV2 pep1jhf and rAAV2 pep1osy via qPCR. A total amount of 1×10¹¹ viral genomes was injected systemically and after 48h incubation organs were extracted from chicken embryos and DNA was isolated before qPCR analysis was performed. Box-whisker diagrams were created using Origin2019 and represent overall replicates (rAAV2 pep1jhf: 7; rAAV2 pep1osy: 11 replicates)..

Besides analysis of genomic copies in the embryonic organs, the transduction of the tumor tissue is of great interest. The mammalian epidermoid carcinoma cell line A431 was transplanted in the CAM and during the incubation time of 96 h a solid tumor was formed. This model was used to evaluate the vectors properties in an in vivo system. Again the genomic copy number in the tumor tissues has been determined in qPCR. A comparison between wild-type rAAV2 and rAAV9 and the re-targeted viral vectors rAAV2 pep1jhf and rAAV2 pep1osy is given in Figure 28. The buffer control as well as wild-type rAAV2 and rAAV2 pep1osy show nearly no transduction at all.

rAAV2 wt can only rely on the natural tropism via the primary receptor HSPG. Cell culture exper-iments showed that in general rAAV2 wild-type is able to transduce A431 cells. In context of the