Design basis of the peptides

Cells were transfected with pEGFP-C1 and addition of similar concentration of L2 protein to the complexes. It was found, that HPV L2 protein increased transfected cell population by approximately 20% in both, CR2 and CR3 of NF51 and reaching 90% in case of CR3 (Figure 9). This result is especially impressive for applying L2 protein in therapeutic pro-tein production.

One of the important parameters for biological applications, cytotoxicity of the L2 protein, was measured after transfection. Results show that L2 protein is not toxic for cells and can be an efficient tool for in vivo applications, as well as for therapeutic protein production (Figure 10).

NF51 CR2 NF51 CR3 Untreated pDNA 0

20 40 60 80

100 0M HPV L2 0.125 M HPV L2

Treatment group

Viability (%)

Figure 10. Viability of transfected cells after L2 protein treatment. CHO cells were transfected with non-covalently formed NF51/pDNA NPs and HPV L2 protein. Plas-mid and untreated cells were used as negative controls. L2 protein does not affect morphology and granularity of cells.

3.2.2 Design basis of the peptides

The promising results with L2 protein inspired the design of shorter peptides derived from L2 protein. As explained in the literature review section of the thesis, L2 has different do-mains with functions responsible for cellular uptake and endosomal escape (Pereira et al.

2009). Three different approaches were applied for peptide design.

For the first screening of peptide design, specific regions from the HPV 16 L2 protein were chosen that have been related to either cellular contacts or endosomal escape functions (Figure 11).

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MRHKRSAKRTKRASATQLYKTCKQAGTCPPDIIPKVEGK- TIAEQILQYGSMGVFFGGLGIGTGSGTGGRTGYIPLGTRPPTATDTLAPVRP- PLTVDPVGPSDPSIVSLVEETSFIDAGAPTSVPSIPPDVSGFSITTSTDTTPAILD- INNTVTTVTTHNNPTFTDPSVLQPPTPAETGGHFTLSSSTISTHNYEEIPMDTFIVST-

NPNTVTSSTPIPGSRPVARLGLYSRTTQQVKVVD-

PAFVTTPTKLITYDNPAYEGIDVDNTLYFSSNDNSINIAPDPDFLDI- VALHRPALTSRRTGIRYSRIGNKQTLRTRSGKSIGAKVHYYYDLSTIDPAEEIEL-

QTITPSTYTTTSHAASPTSINNGLYDIYADD- FITDTSTTPVPSVPSTSLSGYIPANTTIPFGGAYNIPLVSGPDIPINITDQAPSLIPIVPG-SPQYTIIADAGDFYLHPSYYMLRKRRKRLPYFFSDVSLAA

Figure 11. HPV16 L2 sequence used for peptide selection. With marked HPV16 L2 14-46, HPV16 L2 246-265 and HPV16 L2 441-473 chosen for this work.

>sp|P03107|VL2_HPV16 Minor capsid protein L2 OS=Human papillomavirus type 16 OX=333760 GN=L2 PE=1 SV=1.

The HPV16 L2441-473 peptide (P1) is derived from the L2 region w/o modifications. In the L2 protein sequence aa 456-462 is a nuclear localisation signal (NLS) (Wang and Roden 2013), aa 456-461 covers a dynein interacting domain (Florin et al. 2006), DNA binding site in aa 454-462 (Bousarghin et al. 2003), part of L1 binding site in aa 412-455 (Finnen et al. 2003), and aa 462-472 is a NES (nuclear export signal) (Mamoor et al. 2012). This also includes a part of a membrane destabilising peptide in aa 454-500 found in PV (Kämper et al. 2006). The arginine-rich part was discovered to possess similarities with CPPs and it probably protrudes through endosomal membrane allowing interactions with cytoplasmic retromer cargo, which mediates the trans-Golgi delivery of PV capsid (Popa et al. 2015). In the sequence, the dynein interaction site and the transmembrane domain capa-ble of penetrating membranes are in close contact, and it has been suggested that possibly L2 could interact with dynein through a vesicular membrane while trafficking to subse-quent compartments during infection (Schneider et al. 2011), (Florin et al. 2006, Kämper et al. 2006). The aim of including this sequence was to increase cellular uptake of CPP/pDNA complexes.

The HPV16 L2246-265 peptide (P2) is derived from L2 region, which has been shown to in-clude Sortin Nexin 17 (SNX17) binding site at aa 245-257 (Bergant Marušič et al. 2012) and has been related to both virion trafficking (Pim et al. 2015) and trafficking

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L2/pseudogenome complex from late endosome/lysosome (Bergant Marušič et al. 2012).

The SNX17 is binding to a highly conserved binding motif (NPAY) and when SNX17/NPAY interact, the trafficking through endosomal compartments might be delayed and SNX17 mediates the endosomal escape from late endosomes preventing the degrada-tion in lysosomes. SNX17 was also found to enhance the stability of L2 by delaying the trafficking in the endosomal pathway (Bergant Marušič et al. 2012). The aim of including this part of the sequence was to alter the trafficking pathway used by NF51/pDNA com-plexes.

HPV16 L214-46peptide (P3) is derived from L2 N-terminal part and from a multifunctional domain. In the L2 protein aa 13-31 is a receptor binding site able to bind to the cell surface (Roden et al. 2000, Yang et al. 2003a), aa 25-45 is actin binding site (Yang et al. 2003b), (Bronnimann et al. 2013) and aa 13-31 and 35-51 are exposed on the surface (Yang et al.

2003a), (Kawana et al. 1998). The peptide is downstream from two consensus furin cleav-age sites, Arg5 (2RHKR5) and Arg12 (9RTKR12), and for efficient infection the cleavcleav-age at Arg12 is crucial (Bronnimann et al. 2016).

For the second round of the peptide design, two strategies were chosen: a) predicted CPP, based on the C-terminal region of L2 (P1), and b) predicted CPP, based on fused parts from P1 and P2. For the first, predictions were based on HPV16 or HPV18 L2 protein C-terminal region, and screened these for predicted CPPs. The P4 and P5 peptide sequences were designed based on HPV16 or HPV18 L2 protein C-terminal region. While designing peptides, program developed for cell-penetrating peptide prediction (Hällbrink) was used to predict if designed peptide sequence was a potential CPP. The peptides include a CPP sequence (Zhang et al. 2018).

Secondly, fusing sections from HPV16 L2246-265 and HPV16 L2441-473, predicted as CPP.

The P6 and P7 were designed based on sections from HPV16 L2246-265 (NPAYregion need-ed for SNX17 binding and trafficking) and HPV16 L2441-473 (NLS, DNA interacting site, dynein interacting domain, CPP), fused and predicted as CPP.

The stability of the nanoparticles, especially when non-covalent complexation strategy is used, increases with the addition of fatty acid moieties to the peptides (Pärnaste et al.

2017). P1 – P5 peptides, were also synthesised with fatty acid modification. As there was no significant increase of efficacy or observed decrease, P6 and P7 peptides with C18 were not included (Figure 12).

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Peptide Sequence (C-terminally amidated) Origin

Peptides derived from the protein sequence without extensive modifications

P1 ADAGDFYLHPSYYMLRKRRKRLPYFFSDVSLAA HPV16 L2

P2 PTKLITYDNPAYEGIDVDNT HPV16 L2

P3 ASATQLYKTCKQAGTCPPDIIPKVEGKTIAEQIL HPV16 L2

Peptides derived from L2 protein sequence C-terminal region, predicted as CPP, modi-fied

P4 FLYALALAALRKRRKLAA HPV16 L2

P5 YYLALALAALKKRKRLAA HPV18 L2

Peptides derived from fusing sections of HPV16 L2246-265 and HPV16 L2441-473, predicted as CPP

P6 LITYDNPAYEFYLHPSYYMLRKRRKR HPV16 L2

P7 NPAYEGIDFYYMLRKRRKR HPV16 L2

Figure 12. Peptide sequences synthesised from HPV16 L2 protein.