Improving AAV vectors for tumor therapy

Gene therapy using rAAVs was shown to be promising also in the field of cancer research. Therapy of cancer with viral vectors requires a selective transduction of the tumor cells. The natural sero-types of AAVs offer the ability to transduce specific tissues via their unique tropism. A problem in

9 therapy with natural serotypes is the high immunoprevalence of the population.⁹ Studies show that half of the population has already come into contact with AAV2 and has thus been able to produce anti-AAV2 neutralizing antibodies.⁴⁹ Another problem in the treatment of cancer is the biodistri-bution in tissue as well as the targeted transduction of tumor cells. Overcoming of these strong limitations was pursued with various approaches.

Delivery and entry of target cells is induced via the amino acid sequence that defines the capsid shell. Solving the crystal structure provided necessary information on the capsid surface and the exposure of amino acids to the surrounding. Two general methods were applied to change AAVs tropism: rational design of targeting motifs and directed evolution of the capsid proteins.

Directed evolution of AAVs capsid proteins does not require a good knowledge of the capsid struc-ture and transduction mechanisms. In 2003, Müller et al. showed the feasibility of an in vitro ran-dom peptide library system for AAV2.⁵⁰ An approach by Michelfelder et al. also used a random peptide library displayed on the viral capsid in in vitro.⁵¹ The selected library-derived rAAVs trans-duced tumors in vivo.

For rational engineering of the capsid, it must be known at which surface-exposed positions of the VP protein integrations can be carried out without loss of productivity. In the past several groups determined possible insertion sites. For AAV2 two groups have demonstrated that integration of peptide ligands in VP proteins at residue positions 46, 115, 139, 161, 261, 381, 447, 459, 534, 573, 584, 587, and 588 did not interfere with capsid assembly (Figure 3A).^52,53 The targeting peptide to be integrated into the capsid should be structure-independent and not too large to avoid destabili-zation of the capsid shell.⁵⁴ Retargeting towards a new target molecule requires the neutralization of the natural tropism. Main interaction between viral particles and primary receptors was described already for a few serotypes. As the best characterized serotype AAV2 all amino acids interacting with HSPG are well known and mutations in the two arginine residues R585 and R588 allow for strong reduction of transduction efficiency.⁵⁵ For AAV6 both residues are missing and thus not contributing to the interaction with HSPG. Here a lysine residue mediates the interaction and it was shown that a K531E mutation impairs affinity towards HSPG.⁵⁶ The amino acids inducing the in-teraction with the secondary receptor EGFR are not characterized yet. The primary receptor N-linked galactose in AAV9 is strongly N-linked to two amino acids N272 and W503 that are known to be important for binding.³⁷

Integration into the capsid can be directly or indirectly targeting a tumor cell specific feature. In direct approaches a peptide ligand is able to bind a cell-specific target, while in indirect targeting approaches, the interaction with the target cell is mediated via an associated molecule, which is bound to the capsid surface. Indirect targeting was described in the past for an rAAV2 that present a minimal immunoglobin G (IgG) binding domain Z34C in amino acid position 587 (Figure 3B).⁵⁷ rAAVs were loaded with different antibodies and specific transduction of human hematopoietic

10

cell lines was observed. Integration of motifs also allows for site-specific bioorthogonal labelling of rAAV particles. Previous work showed that integration of the recognition motif for the formyl-glycine-generating enzyme (FGE) was possible at amino acid position 587 and allowed for covalent conjugation of the resulting aldehyde either with Alexa488 hydrazide or amine-functionalized gold particles (Figure 3B).⁵⁸

Direct targeting for rAAV2 was shown with different peptide integrations in position 587 but also with VP2 N-terminal fusions. RGD peptides have been incorporated into surface-exposed VP ar-eas.⁵³ It was shown that cells were transduced independently from the natural HSPG motif. The N-terminal fusion of even whole proteins to the VP2 protein was demonstrated by different groups.^59,60 In both approaches a four-plasmid system is required where the VP2 fusion protein is delivered separately from VP1 and VP3 (Figure 3C).

Figure 3: Strategies for rAAV retargeting. (A) Single amino acid mutations can be genetically introduced. (B) Site-specific integration is tolerated at various sites of the VP proteins and enables for further non-genetic modifications of the capsid. Absorption of IgG molecules to integrated Z34C domains was shown previously to results in biologically active vectors. Integration of motifs also allows for further biorthogonal labelling, e.g. using the formylglycine gen-erating enzyme (FGE) to generate an aldehyde available for covalent conjugation of amine-functionalized gold par-ticles (Au). (C) Integration of motifs or fusion proteins does not necessarily result in fully-modified capsids. Genera-tion of so-called mosaic viral vectors remains possible with an alteraGenera-tion in the plasmid system.^59,60

All systems described were developed for serotype 2 but since the capsid similarity between sero-types is high, some groups also established incorporation of peptides into other serosero-types. From random peptide libraries selected peptides have been transferred from serotype 2 to serotype 8 and 9.⁶¹ Here it was shown, that not only the peptide sequence optimized for AAV2 determines the transduction ability in vivo but also the overall capsid contributes to the tropism. A different ap-proach relying on retargeting using RGD peptides was shown in AAV6.⁶²

cap(VP 123) cap(VP 123) ^motif

Amino acid mutation

Absorption of molecules,

e.g. IgG

Bioorthogonal Labelling, e.g. using FGE

VP2 cap (VP13)

Au Au

Au Au

Au Au Au

Au Non-genetic

modification Integration of motifs

motif Mosaic capsids

A B C

11 In addition to the specific targeting of the cell by the viral capsid, tumor therapy can also use other properties that e.g. antibodies do not have. The viral particles do not introduce any active substance into the cells. Instead, only the DNA is specifically delivered under the control of a promoter. The expression of the target protein can be specifically activated in tumor cells in various ways. One example is the promoter of the C-X-C chemokine receptor type 4 (CXCR4). For this promoter it had been shown in the context of AAV2 that expression of the transgene was only achieved in tumor cells.⁶³ A second approach is the use of anti microRNAs (miRNA). The Let-7 family has twelve known members that target the same mRNA sequenes.⁶⁴ Members of this family are known to accumulate in differentiated cells, but were shown to be downregulated in cancer cells by mech-anism that are not fully understood.⁶⁵ After transduction into healthy cells, the mRNA of the deliv-ered transgene is degraded by binding the anti-Let-7 miRNA, while translation can take place in tumor cells.⁶⁶ Specific targeting combining both approaches can be summarized under the term virus-directed enzyme prodrug therapy (VDEPT).⁶⁷ This emerging strategy in the treatment of can-cer allows for direct targeting of cells via tumor specific features. By delivering of an enzyme, a prodrug is activated into a cytotoxic compound that finally leads to apoptosis of the cancer cell. In the past, several targets of cancer cells have been identified and used for therapy. The epidermal growth factor receptor (EGFR) is one example for a validated tumor target.⁶⁸