# Bioorthogonal Chemical Engineering of rAAV Capsid: Advancing Gene Therapy Targeting Using Proteins

Maia Marchand, Sébastien Depienne, Mohammed Bouzelha, Karine Pavageau, Roxane Peumery, Denis Loquet, Dimitri Alvarez-Dorta, Mickaël Guilbaud, Mikaël Croyal, Aurelien Dupont, et al.

► To cite this version:

Maia Marchand, Sébastien Depienne, Mohammed Bouzelha, Karine Pavageau, Roxane Peumery, et al. Bioorthogonal Chemical Engineering of rAAV Capsid: Advancing Gene Therapy Targeting Using Proteins. Bioconjugate Chemistry, 2025, 36 (3), pp.521-530. 10.1021/acs.bioconjchem.4c00580. hal-04985237

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Bioorthogonal chemical engineering of rAAV capsid: Advancing gene therapy targeting using proteins

Maia Marchand,<sup>1,2,*</sup> Sébastien Depienne,<sup>2</sup> Mohammed Bouzelha,<sup>1</sup> Karine Pavageau,<sup>1</sup> Roxane Peumery,<sup>2</sup> Denis Loquet,<sup>2</sup> Dimitri Alvarez-Dorta,<sup>2,3</sup> Mickaël Guilbaud,<sup>1</sup> Mikaël Croyal,<sup>4,5</sup> Aurélien Dupont,<sup>6</sup> Oumeya Adjali,<sup>1</sup> Sébastien G. Gouin,<sup>2</sup> David Deniaud<sup>2,*</sup> & Mathieu Mével<sup>1,2,*</sup>.

<sup>1</sup>Nantes Université, TaRGeT, Translational Research for Gene Therapies, CHU Nantes, INSERM, UMR 1089, F-44000 Nantes, France.

<sup>2</sup>Nantes Université, CNRS, CEISAM, UMR 6230, F-44000 Nantes, France.

<sup>3</sup>Capacités SAS, Nantes, France.

<sup>4</sup>Nantes Université, CNRS, INSERM, Institut du thorax, F-44000 Nantes, France.

<sup>5</sup>Nantes Université, CHU Nantes, Inserm, CNRS, SFR Santé, Inserm UMS 016, CNRS UMS 3556, F-44000 Nantes, France.

<sup>6</sup>CNRS, INSERM, BIOSIT_UAR 3480, Univ Rennes, Inserm 018, F-35000 Rennes, France.

*Corresponding authors:

maia.marchand@univ-nantes.fr
david.deniaud@univ-nantes.fr
mathieu.mevel@univ-nantes.fr

ABSTRACT

We report the chemical conjugation of recombinant Adeno Associated Virus (rAAV) capsid with various functionalities, including proteins, using a bioorthogonal strategy. rAAVs were azido-coated or DBCO-coated by chemically modifying lysine or tyrosine residues. Lysine residues were modified using a phenyl isothiocyanate anchor, and tyrosine residues using either an aryldiazonium salt or a *N*-methyl luminol derivative. We demonstrate anchor-dependent labelling levels, as observed with biochemical assays and mass spectrometry. Strain-promoted azide-alkyne cycloaddition (SPAAC) was then implemented and evaluated on the rAAV to append functionalities such as fluorescein, biotin and carbohydrates to the azido-coated capsids. We confirmed the efficiency of the bioorthogonal reaction and observed a stronger reactivity with dibenzylcyclooctyne (DBCO) compared to bicyclononyne (BCN). The optimized SPAAC reaction was finally used to label the viral vectors with two relevant nanobodies targeting specific immune cell receptors (CD62L and CD45). *In vitro* transduction assays conducted with one rAAV-nanobody conjugate demonstrated the promising targeting properties of these chemically modified vectors. Thus, we anticipate that this strategy will positively impact the field of rAAV capsid engineering and contribute in tissue-specific targeting for the optimisation of gene therapy treatments.
## KEYWORDS

rAAV, SPAAC, bioorthogonal click chemistry, bioconjugation, nanobody, gene therapy.

## INTRODUCTION

In recent years, recombinant Adeno Associated Viruses (rAAVs) have emerged as one of the most promising vector candidates in clinical studies for *in vivo* gene therapy.

This is primarily attributed to their ability to transduce a diverse array of cells and tissues with persistent transgene expression, as well as a good safety profile¹. Patients suffering from metabolic to retinal disorders are now treated with rAAVs using a single injection protocol²⁻⁴.

Thirteen AAV serotypes were originally described in the literature¹,⁵. For each one, preferentially transduced tissues were identified⁶, such as rAAV9 which demonstrated interesting gene delivery capabilities in the brain⁷. However, these tropisms remain too broad, particularly because a significant proportion of rAAV particles end up in the liver, resulting in poor tissue specificity, off-target expression, and reduced efficiency. To address this issue, high doses of vectors need to be administered to the patient, which enhances the risk of immune toxicity⁸ and makes large-scale production of vectors challenging,⁹ leading to expensive treatments.

As a key approach to redirect tropism and improve the therapeutic index, the design of the viral capsid is extensively explored. Among the strategies, chimeric vectors have been produced by mixing viral proteins from different serotypes to take advantage of their distinct characteristics¹⁰,¹¹. Additionally, larger rAAV libraries were generated through directed evolution approaches, shuffling methods or peptide display techniques¹²,¹³. A prominent example has been the rAAV-PHP.eB capsid, which demonstrated an enhanced transduction in the central nervous system¹⁴. Nevertheless, without rational design, such strategies rely on screening experiments in *in vivo* models, which may not always accurately represent the actual rAAV biodistribution in human¹⁵. More rational approaches have also been explored including specific genetic insertion¹⁶⁻¹⁸. Non-natural amino acids bearing specific anchors were incorporated into VP viral protein (VP) sequences to further append relevant small molecules or oligonucleotides using bioorthogonal reactions¹⁹⁻²³. Additionally, the highly specific targeting properties of proteins and peptides have also been exploited. For instance, a noteworthy construction involving DARPs fused to the VP2 N-terminus has been described as a relevant strategy to redirect the rAAV toward HER2/neu⁺ cells²⁴. Genetic engineering continues to provide precise control over the site and degree of modification, which is of significant importance for the rAAV properties. However, the production process needs adaptation for each case, and the vectors often suffer from low titers²⁵.
Alternatively, recent investigations, including our previous works, have explored the direct chemical modification of rAAV vectors. Residues such as lysine (K) or tyrosine (Y) composing the proteins of the AAV capsid, have been bioconjugated with targeting or imaging small molecules (e.g. carbohydrates, radionuclides, fluorophores) resulting in a promising improvement or monitoring of the transduction of selected tissues<sup>26-28</sup>. However, the conjugation of complex structures, particularly proteins, inherently cannot be explored using this direct bioconjugation strategy.

In this study, we demonstrated and optimized a direct chemical sequence featuring biorthogonality to access rAAV-protein conjugates as a faster and easier alternative to genetic manipulations. We first achieved the obtention of an azido-coated rAAV using chemoselective bioconjugation on K or Y residues, to evaluate and compare potential efficiency differences. Phenyl isothiocyanate and aryl diazonium salt were the bioconjugation anchors selected for K and Y modification, respectively, owing to their non-disruptive and efficient modification of rAAVs<sup>26,28</sup>. Additionally, we explored the functionalization of rAAVs with a *N*-methyl luminol (NML) derivative using the Y-targeting click-electrochemistry strategy recently reported by our group for viral and cell surfaces labelling<sup>29</sup>. Azido-coated rAAVs were successfully obtained in all cases, with labelling levels dependent on the bioconjugation strategy. Subsequently, post-functionalization using the bioorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC)<sup>30</sup> was illustrated and optimized with cyclooctyne-derivatized small molecules (fluorescein, biotin, *N*-acetylgalactosamine). Interestingly, DBCO (dibenzylcyclooctyne) exhibited a significantly higher reactivity than BCN (bicyclononyne) in our model. On the other hand, the chemical bioconjugation of rAAV with the valuable DBCO click handle was successfully conducted, which, to the best of our knowledge, has never been reported before. Subsequently, SPAAC post-functionalization using a synthetic azido-derivatized fluorescent probe was similarly proven at the capsid scale, highlighting the possibility to easily and rapidly label rAAVs with a broad range of azido-tagged functionalities. With the optimized bioorthogonal sequence in hands, two biologically relevant nanobodies anti-CD45 and anti-CD62L (targeting specific immune cells markers<sup>31,32</sup>) were successfully covalently linked to the viral capsid to afford rAAV-nanobodies conjugates with conserved transduction capacity in HeLaT cells. More interestingly, the transduction in the presence of the cell-binding inhibitor heparin was partially maintained in the CD45-expressing cell line Jurkat when rAAV was labelled with the anti-CD45 moiety, highlighting the therapeutic prospect of the bioorthogonal strategy. These promising results contribute to the advancement of next-generation capsid engineering, paving the way for tissue-targeted gene therapy.
RESULTS AND DISCUSSIONS

Obtention of azido-rAAV2

Our investigations started with the chemical labelling of the rAAV capsid with azido moieties.
Considering the potential impact of amino acid selection and bioconjugation anchors on both
the efficiency and level of functionalization (affecting the resulting vector profiles²⁶,²⁸), we
compared several strategies. Three distinct anchors were designed, based on previous reports
where rAAV labelling with carbohydrates has been successfully achieved on K and Y
residues²⁹,³³. The azido moiety was associated with a phenyl isothiocyanate for K modification,
an aryl diazonium salt for the Y one and a *N*-methyl luminol to explore electrochemical Y-
conjugation. Their synthesis is outlined in Scheme 1. We first carried out a three-step synthetic
route to obtain the azido-amino linker **4** using triethylene glycol. After tosylation of the two
alcohol groups of compound **1**, and substitution with sodium azide, we performed the
Staudinger reduction of only one of the azido moieties thanks to biphasic conditions. To obtain
the K targeting anchor **Lys-N<sub>3</sub>**, a one-step *N*-functionalization of the amine **4** with bitoscanate
was carried out. To synthesize the azido-aniline derivative **6**, the same intermediate **4** was
used to perform a peptide coupling with 4-aminobenzoic acid protected with a BOC group,
which was removed in an additional deprotection step with TFA. This molecule **6** served as the
substrate for diazonium-activation (**Tyr-N<sub>3</sub>**) just before the bioconjugation reaction.
Additionally, we carried out a previously reported four-step synthesis²⁹ to obtain from **7** the
azido-NML derivative **eTyr-N<sub>3</sub>**, the anchor being electro-oxidized to label tyrosine.

<figure><img src="image_2.png" /><figcaption>**Scheme 1:** Synthesis of the bioconjugation anchors. (a) **Lys-N<sub>3</sub>** for K functionalization, **Tyr-N<sub>3</sub>** for chemical Y functionalization, (b) **eTyr-N<sub>3</sub>** for Y electrochemical functionalization. (i) TsCl, Et<sub>3</sub>N, DCM, RT, 18 h (69 %); (ii) According Budin *et al.* (2010)<sup>34</sup>: NaN<sub>3</sub>, DMF, 70°C, 18 h (quant.); (iii) According to Goswami *et al.* (2013)<sup>35</sup>: PPh<sub>3</sub>, Et<sub>2</sub>O, 5 % HCl, RT, 20 h (84 %); (iv) DMF, RT, 4 h (88 %); (v) HBTU, DMAP, DMF, RT, 3 h (80 %); (vi) TFA, DCM then basic resin, RT, 4 h (96 %); (vii) HBF<sub>4</sub>, tBuONO, dPBS, RT, 5 min (quant.); (viii) 4-step synthesis by Depienne *et al.* (2021)<sup>36</sup>.</figcaption></figure>
For the bioconjugation step, we selected rAAV2 vectors, a highly studied and characterized serotype⁵. Conditions using 1E5 equivalents of **Tyr-N₃** and **Lys-N₃** (Fig. 1.a) and a concentration of 0.5 mM of **eTyr-N₃** were chosen according to the reported reactivity of the corresponding anchors (Fig. 1.b). The bioconjugation reactions were performed as follows: **Lys-N₃** was directly co-incubated with 2E12 vg of rAAV2 in 1 mL of TBS pH 9.3 for 4 h; **Tyr-N₃** was obtained from 6 by the action of hexafluoroborate (HBF₄) followed by *tert*-butyl nitrite (*tBuONO*), added to a solution containing 2E12 vg of rAAV2 in 560 µL of dPBS and stirred for 1 h; **eTyr-N₃** was electro-oxidized for 30 min at 750 mV vs Ag/AgCl in a 1 mL solution of 1E12 vg of rAAV2 according to the experimental setup presented in Fig. S1. After dialysis and titer quantification, the resulting vectors were characterized using various analytical techniques. The A20 antibody was employed in the dot blot assay to discern any alterations in the capsid 3D structure, confirming that the conjugation step did not induce structural modifications (Fig. 1.c). To validate the azido labelling of rAAVs, we substituted the antibody in the dot blot analysis with fluorescein conjugated to the BCN structure (**BCN-F**). For any azido moieties present on viral proteins, a SPAAC reaction would occur directly on the nitrocellulose membrane, resulting in a fluorescent signal (Fig. 1.c). Satisfyingly, we confirmed the functionalization of the three azido-conjugated rAAV2. A strategy-dependent level of labelling was observed with a notably stronger fluorescence signal with lysine (**AAV2-Lys-N₃**) and electro-tyrosine modification (**AAV2-eTyr-N₃**) as compared to chemical tyrosine modification (**AAV2-Tyr-N₃**). The effective bioconjugation of **AAV2-Lys-N₃** and **AAV2-eTyr-N₃** was further supported by mass spectrometry analyses. Azido-labelled rAAV2 were injected into an UPLC column without any denaturation treatment. We focused on the mass spectrum corresponding to VP3, the smallest and most abundant of the three viral proteins (VP1-3) composing the capsid³⁷. For **AAV2-Lys-N₃**, the deconvoluted mass spectrometry profile of VP3 confirmed the presence of the protein with zero, one, or two covalently conjugated anchors (Fig. 1.e). More specifically, considering the ratio of modified to non-modified proteins, as well as the number of VP3 proteins in viral particles, we can estimate that approximately 30 azido moieties are exposed on the viral surface. Electro-conjugation resulted in a higher degree of functionalization, since the native VP3 protein was not detectable. On average, proteins displayed four modifications, resulting in approximately 160 azido groups exposed at the AAV surface (Fig. 1.f). Unfortunately, we could not confirm the labelling of **AAV2-Tyr-N₃** by mass spectrometry, likely due to a lesser number of modified proteins.

To confirm that the labelled vectors conserved their functional integrity, we evaluated their infectivity on HeLa cells. Cells were co-incubated with azido-functionalized rAAV2 carrying an Enhanced Green Fluorescent Protein (eGFP) reporter gene expression cassette under the control of a ubiquitous promoter at a multiplicity of infection (MOI) of 1E3, and the percentage of fluorescent cells was measured after 48 hours by flow cytometry (Fig. 1.d). No significant
loss of transduction was observed, indicating that neither the bioconjugation conditions nor the azido coating affected GFP expression.
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>
**Figure 1: Bioconjugation of rAAV2 with azido derivatives and characterization of the obtained vectors.** a) rAAV2 bioconjugation with **Lys-N<sub>3</sub>** or **Tyr-N<sub>3</sub>** using 1<sup>E5</sup> equiv. in aqueous buffers. b) rAAV2 electro-bioconjugation after an *in situ* electro-oxidation (750 mV vs Ag/AgCl) of *eTyr-N<sub>3</sub>* for 30 min at 0.5 mM in dPBS. c) Dot Blot analysis of viral vectors to detect (up lane, 2<sup>E10</sup> vg) the assembled capsid using the A20 antibody followed by secondary revelation using an anti-mouse-HRP antibody or to detect (down lane, 3<sup>E10</sup> vg) the azido moieties using **BCN-F**. d) Evaluation of the transduction efficiency in HeLaT of modified rAAV2 carrying eGFP reporter gene expression cassette under the control of a ubiquitous promoter. GFP-positive HeLaT quantification by flow cytometry 48 h after infection at a MOI of 1<sup>E3</sup> (data are shown as mean ± SD of three distinct samples (n=3)). e) and f) Analysis by mass spectrometry of rAAV2-Lys-N<sub>3</sub> and rAAV2-eTyr-N<sub>3</sub>. Vectors were analyzed without any denaturation step. Mass profiles were obtained after migration on an UPLC column and deconvolution of the VP3 corresponding peak (MW (Lys-N<sub>3</sub>) = 366.46 g/mol and MW (eTyr-N<sub>3</sub>) = 261.24 g/mol).

**Comparison of the reactivity of two cyclooctynes**

Various cyclooctynes have been reported in the literature for their efficient reaction with azido moieties in a bioorthogonal manner³⁸. In this study, we focused on BCN and DBCO to explore the post-functionalization of azido-labelled rAAV2 using the SPAAC reaction. Their reactivity was assessed on **rAAV2-Lys-N<sub>3</sub>** (using a solution at 1<sup>E12</sup> vg/mL) over a period of one hour with 10 µM of a fluorescent cyclooctyne in each case (Fig. 2.a). Dot blot analysis, based on direct fluorescence measurement, showed a stronger signal with the DBCO structure compared to BCN while no modification of the 3D structure was observed with A20 detection (Fig. 2.b). The difference in post-functionalization was further supported by SDS-PAGE analysis. The three specific viral proteins constituting the capsid (VP1, VP2, and VP3) were unambiguously modified with **DBCO-F**, as three distinct fluorescent bands corresponding to the molecular weights of these proteins were observed. These bands were clearly not detected with the **BCN** scaffold (Fig. 2.c). It is noteworthy that two VP3 populations were greatly separated on the silver staining of DBCO modified vectors (red arrow on Fig. 2.d). We can hypothesize that the larger one may correspond to a modification of the VP3 unit, providing additional evidence of the successful SPAAC reaction. Given the observed difference in reactivity, DBCO derivatives were chosen for the post-functionalization of azido-labelled rAAV2, and the fluorescein-coating of **rAAV2-Tyr-N<sub>3</sub>** and **rAAV2-eTyr-N<sub>3</sub>** was explored in Fig. S13.
<figure><img src="image_9.png" /><figcaption>**Figure 2:** Comparison of the efficiency of BCN and DBCO cyclooctynes to post-functionalize azido-vectors.
a) SPAAC reaction for rAAV2-Lys-N₃ post-functionalization using BCN-F and DBCO-F at 10 µM for 1 h.
b) Dot Blot analysis of viral vectors to detect (up lane, 1.5E10 vg) the covalent linkage of fluorescein by direct fluorescence measurement or to detect (down lane, 2E10 vg) the assembled capsid using the A20 antibody followed by secondary revelation using an anti-mouse-HRP antibody.
c) Detection of fluorescein on capsid proteins with direct fluorescence measurement after denaturation and migration by SDS-PAGE of modified or non-modified rAAV2 (1.5E10 vg).
d) Silver staining of capsid viral proteins after denaturation and SDS-PAGE migration of modified or non-modified rAAV2 (1.5E10 vg).</figcaption></figure>

**Azido-rAAV2 post-functionalization with DBCO-derivatized small molecules**

To further demonstrate the rapid and efficient access to a variety of functionalized rAAV2, we expanded the SPAAC reaction to include biotin and GalNAc derivatives, using 50 µM of **DBCO-B** and **DBCO-G** respectively (structures and syntheses are provided in the Supplementary Information, Fig. 3.a). Biotin is widely recognized in the literature as an effective molecular label³⁹, while GalNAc serves as hepatocyte targeting moiety⁴⁰.

Each modification was specifically studied after 4 h using dot blot assays. Streptavidin was employed to detect biotin and the soybean lectin specific to the GalNAc carbohydrate was used. We successfully confirmed the SPAAC reaction with the two functionalities on the three azido-coated rAAV2 (Fig. 3.b). The results corroborated our previous findings. Notably, a higher degree of post-modification was obtained from the more azido-labelled **AAV2-Lys-N₃** and **AAV2-eTyr-N₃**, compared to **AAV2-Tyr-N₃**. Despite a very low concentration of rAAV2
(C<sub>AAV</sub> is approximately 1 nM), a higher number of azido groups on the capsid resulted in a higher degree of post-functionalization. This observation highlights the interesting opportunity for grafting modulation. Importantly, unmodified rAAV2 were reacted under the same conditions with the derivatized DBCO which showed a total absence of signals detection (Fig. S14.a). rAAV2 vectors were also analyzed with a A20 dot blot assay, and no modifications in capsid integrities were noticed (Fig. 3.b, lane 2). Functionalized viral proteins were additionally visualized by SDS-PAGE followed by silver staining (Fig. 3.c). Consistent with previous observations, this assay demonstrated that the electrochemically-labelled AAV was the most highly post-functionalized vector in all cases. For rAAV2-eTyr-B, the modification may even be too drastic as it is difficult to clearly identified which band corresponds to which protein. On the contrary, despite a similar working concentration (50 µM for DBCO-B and DBCO-G), we can clearly identify the viral proteins on rAAV2-eTyr-G SDS-PAGE profile, which can indicate a difference in reactivity between the DBCO constructs. Furthermore, the profile particularly echoes the mass spectrometry analysis presented in Fig. 1.f. Indeed, three distinct levels of VP3 modification could be observed (red arrows in Fig. 3.c), providing an evidence that poly-modifications of the proteins is possible.

Gratifyingly, in HeLa cells, almost all functionalized vectors induced GFP production at levels comparable to unmodified rAAV2 (Fig. S14.c). Unsurprisingly, the exception was **rAAV2-eTyr-B**, which showed lower transduction efficacy. Considering its SDS-PAGE profiles, the observed differences in transduction could be attributed to a too high degree of functionalization of the capsid with crowded and/or hydrophobic structures. Both the duration of SPAAC reaction and the concentration of DBCO derivatives seem to be key factors that can be adjusted to fine-tune capsid engineering. Nevertheless, all the data mentioned underscore the interest of the three obtained azido-labelled AAVs for accessing a variety of post-functionalized vectors.
<figure><img src="image_10.png" /><figcaption>**Figure 3:** SPAAC reaction on azido-rAAV2 with DBCO-derivatized small molecules. a) SPAAC reaction for rAAV2-Lys-N<sub>3</sub>, rAAV2-Tyr-N<sub>3</sub> and rAAV2-eTyr-N<sub>3</sub> using a DBCO-derivatized N-acetylgalactosamine (DBCO-G) and a DBCO-derivatized biotin (DBCO-B). b) Dot Blot analysis of viral vectors to detect (up line) the covalent labelling of the capsids with DBCO derivatives or to detect (down lane, 1<sup>E</sup>10 ng) the assembled capsid using the A20 antibody followed by secondary revelation using an anti-mouse-HRP antibody. GalNAc labelling was detected (2<sup>E</sup>10 ng) using a FITC-conjugated Soybean Agglutinin (SBA) lectin followed by secondary revelation using an anti-FITC-HRP antibody. Biotin labelling was detected (1<sup>E</sup>10 ng) with an HRP-conjugated Streptavidin. c) Silver staining of capsid viral proteins after denaturation and SDS-PAGE migration of modified or non-modified rAAV2 (1.5<sup>E</sup>10 ng).</figcaption></figure>

**DBCO vector and post-functionalization with an azido derivative**

DBCO-labelled vectors can also be developed as interesting targets to expand the
scope of post-functionalization. Indeed, azido derivatives can sometimes be more accessible
than their corresponding DBCO counterparts. For instance, non-natural azido amino acids can
be easily incorporated into protein sequences with control over the number and sites of
incorporation, thanks to significant advancements in the field of genetic engineering<sup>41</sup>. DBCO
derivatives can sometimes be difficult to synthesize, and stability and solubility issues may
arise. In such cases, replacing them with an azido group in the synthetic route would represent
an interesting alternative solution. To maximize the potential of chemical conjugation for rAAV
engineering, we explored reversing the roles of the two click partners and investigated the
possibility of obtaining DBCO-conjugated rAAVs.
Due to solubility issues of DBCO associated with the lysine conjugating moiety, we only investigated the labelling with the tyrosine chemical anchor. The desired aniline **12** was synthesized in a single step of peptide coupling (Fig. 4.a). The corresponding diazonium salt **Tyr-DBCO** was obtained in DMSO just before the conjugation step, and 1E5 equivalents (in 56 µL of DMSO) were added to the rAAV2 mixture (510 µL of dPBS containing 1E12 vg) for a 1 h stirring (Fig. 4.b). Similar to azido-rAAVs, after titer quantification, we carried out a dot blot analysis using an azido-fluorescein derivative (**N**<sub>3</sub>-**F**) to perform the SPAAC reaction on the nitrocellulose membrane, confirming the presence of the DBCO moiety. As shown in Fig. 4.c, the fluorescent signal clearly demonstrated the covalent coupling of the capsid with the DBCO moiety, without affecting the 3D structure, as indicated by the additional A20 staining. The DBCO vector was characterized by mass spectrometry, and the obtained profile confirmed the bioconjugation of rAAV2. Specifically, we observed that VP3 was partially mono-bioconjugated and estimate that around 20 DBCO are exposed on the capsid (Fig. 4.d).

To validate the post-functionalization of DBCO vectors, we explored the conjugation of **N**<sub>3</sub>-**F** (100 µM). Interestingly, on the silver staining analysis shown in Fig. 4.e, three additional bands were clearly observed above the three viral proteins (red arrows). Combined with the intense fluorescent signal observed in SDS-PAGE analysis with fluorescence revelation in Fig. 4.f, this unequivocally demonstrates the successful and efficient probe labelling. DBCO-coated rAAVs complement the previously described azido vectors, contributing to the development of a broad-spectrum functionalization strategy for rAAV capsid engineering.
<figure><img src="image_11.png" /><figcaption>**Figure 4:** Obtention of DBCO-labelled rAAV2: synthesis of the tyrosine anchor, characterization of the obtained vector and post-functionalization with N₃-F. a) Synthesis of the **Tyr-DBCO** compound. (i) HBTU, DMAP, DMF, RT, 4 h (51%); (ii) HBF₄, tBuONO, DMSO, RT, 5 min (quant.). b) rAAV2 bioconjugation with **Tyr-DBCO** using 1E5 equiv. in dPBS and SPAAC reaction using an azido-derivatized fluorescein (N₃-F) at 100 µM. c) Dot Blot analysis of viral vectors to detect (up lane, 3E10 vg) the DBCO moieties using N₃-F or to detect (down lane, 1E10 vg) the assembled capsid using the A20 antibody followed by secondary revelation using an anti-mouse-HRP antibody. d) Analysis by mass spectrometry of rAAV2-Tyr-DBCO. Vectors were analyzed without any denaturation step. Mass profiles were obtained after migration on an UPLC column and deconvolution of the VP3 corresponding peak (MW (Tyr-DBCO) = 566.64 g/mol). e) Silver staining of capsid viral proteins after denaturation and SDS-PAGE migration of N₃-F modified and non-modified rAAV2 (1.5E10 vg) f) Detection of fluorescein on capsid proteins with direct fluorescence measurement after denaturation and SDS-PAGE migration of fluorescein modified and non-modified rAAV (1.5E10 vg).</figcaption></figure>

rAAV2 post-functionalization with proteins

The effective post functionalization of azido-rAAV2 or DBCO-rAAV2 was initially validated with small molecules. However, larger structures, including biomolecules, can also possess interesting properties, particularly in terms of targeting<sup>42,43</sup>. For instance, nanobodies have been extensively explored due to their size, ease of production, and high specificity, making them powerful tools for tissue-specific addressing<sup>44</sup>. In this study, we explored the possibility of post-functionalizing the azido vectors **rAAV2-Lys-N<sub>3</sub>** and **rAAV2-eTyr-N<sub>3</sub>** with two biologically relevant nanobodies: anti-human CD62L and anti-human CD45. The former interacts with the cell adhesion molecule CD62L which is implicated in immune cell
interactions⁴⁵, while the latter binds to the well-described leukocyte marker CD45⁴⁶. The SPAAC reaction was carried out for 1 or 4 h and a concentration of 15 µM of DBCO-derivatized nanobodies was used (Fig. 5.a). The reaction was performed in the presence of different solutions of azido-rAAV2 at 1E12 vg/mL in dPBS (Fig. 2). Silver staining analysis of rAAV2-Lys-CD62L, rAAV2-eTyr-CD62L, rAAV2-Lys-CD45, and rAAV2-eTyr-CD45 unambiguously revealed a new protein band slightly above 75 kDa (indicated by red arrows in Fig. 5.b). This supports the successful functionalization of the corresponding nanobodies (16 kDa each) onto VP3 (approximately 60 kDa) under SPAAC conditions. To confirm that this “75 kDa” band corresponds to a nanobody-functionalized viral protein, western blot analyses were performed using an antibody specific of the nanobody structure. As depicted in Fig. 4.c, the nanobody anti-CD62L was strongly detected in the “75 kDa” band for both rAAV2-Lys-CD62L and rAAV2-eTyr-CD62L, confirming the effective formation of the nanobody-viral vector conjugate without affecting the size and structure of the nanobody. For anti-CD45, a less intense but visible recognition of the 75 kDa band was observed. Additionally, the viral origin of those same bands was further confirmed by western blot using polyclonal antibodies against VP1, VP2, and VP3 (Fig. S16.a). Importantly, all nanobody-rAAV2 conjugates reproducibly conserved the integrity of their assembled capsid and their infectivity on HeLaT cells (Fig. S16.c). Cryogenic electron microscopy (cryo-TEM), was used to evaluate the effect of nanobody conjugation on capsid geometry. As expected, a hexagonal shape was observed for unmodified rAAV2. Interestingly, outgrowths similar to “spines” were noticed (white arrows in Fig. 5.d) for the conjugated vectors rAAV2-Lys-CD62L and rAAV2-eTyr-CD62L. These specific structures were not detected in images of azido-rAAV2 (Fig. S11.c), strongly indicating the presence of covalently linked nanobodies on the capsid surface. No such outgrowths were clearly observed using the anti-CD45 nanobody, likely due to a lower level of modification (data not shown).
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /><figcaption>**Figure 5:** SPAAC reaction on azido-rAAV2 with two DBCO-derivatized nanobodies. a) SPAAC reaction for rAAV2-Lys-N<sub>3</sub> and rAAV2-eTyr-N<sub>3</sub> using two DBCO-derivatized nanobodies, one targeting CD62L and the other targeting CD45. b) Silver staining of capsid viral proteins after denaturation and SDS-PAGE migration of nanobodies modified and non-modified rAAV2 (1.5<sup>E</sup>10 vg). c) Western blot analysis of nanobodies modified and non-modified rAAV2 to detect nanobodies structures with an anti-VHH-HRP antibody or anti-VHH-FITC antibody followed by secondary revelation using an anti-FITC-HRP antibody (1<sup>E</sup>10 vg). d) Cryo-TEM images of rAAV2 and nanobodies modified rAAV2. Scale bars correspond to 25 nm. One representative image was chosen for each sample.</figcaption></figure>
To complete biochemical characterizations, we investigated the targeting ability of the nanobody-conjugated vectors. We selected the rAAV2-Lys-CD45 vector for transduction assays on the CD45-expressing cell line Jurkat. This cell line was chosen as this marker is highly expressed on its surface (Fig. S17). Having demonstrated that the nanobody used for rAAV labelling was efficiently interacting with this receptor (Fig. S17), Jurkat cells were infected at MOIs of 5E2 and 1E3. Unexpectedly, after 72 h, the number of GFP-positive cells, as observed by flow cytometry, was similar for both rAAV2 and rAAV2-Lys-CD45, suggesting that the labelling did not enhance the transduction of CD45-expressing cells (Fig. 6.a). One hypothesis to explain the obtained results could be that the natural entry pathway of rAAV2 masks the one that can be induced by an interaction between CD45 and the nanobody. The rAAV of serotype 2 is using the primary receptor Heparan Sulfate ProteoGlycan (HSPG) to interact with the cells and enter into the cytoplasm⁴⁷. Thus, to test this hypothesis, the natural entry pathway of rAAV2 was blocked by performing a heparin binding inhibition assay on Jurkat and HeLa cells (a non-expressing CD45 control cell line). In presence of 500 µg/mL of heparin, an almost full inhibition of the Jurkat cells transduction was observed with unmodified rAAV2. Indeed, the transduction efficiency observed without heparin was divided by ten (Fig. 6.b and S18). On the contrary with rAAV2-Lys-CD45, a residual transduction of 25 % was obtained which means that the GFP production could be partially maintained in presence of heparin. These encouraging observations are specific to the CD45 expressing cell line as no significant difference in the percentage of residual transduction was observed between the two vectors in HeLa cells. Furthermore, the results presented in Fig. S19 for rAAV2-Lys-N₃ confirmed that it is not the bioconjugation reaction itself that influences this difference in transduction efficiency.

These preliminary data suggest that, in the absence of heparin, the HSPG pathway is the main entry pathway used by rAAV2-Lys-CD45, and that the interaction between the nanobody and the CD45 marker does not increase it. When the vector cannot interact with HSPG, the presence of the CD45 targeting moiety on the capsid partially maintains the transduction efficiency of the vector, suggesting that a new entry pathway could be used. Interestingly, this hypothesis was supported by the observed decrease in residual transduction when Jurkat cells were pre-incubated with free anti-CD45 nanobodies, which blocked the interaction between the CD45 protein and rAAV2-Lys-CD45, prior to transduction in the presence of heparin (Fig. S20). In the light of the results, mutating arginine residues responsible for the HSPG interaction (R588 and R585) in the rAAV2 sequence ⁴⁸⁻⁵⁰ and performing the bioorthogonal sequence on those specific vectors, with more optimized SPAAC conditions, would represent a promising alternative to develop specific vectors capable of transducing only CD45 expressing cells.
<figure><img src="image_16.png" /><figcaption>**Figure 6: Evaluation of the transduction efficiency of rAAV2-Lys-CD45 in a CD45 expressing cell line.**
a) Evaluation of the transduction efficiency in Jurkat cells of rAAV2 and rAAV2-Lys-CD45 carrying eGFP reporter gene expression cassette under the control of a ubiquitous promoter. GFP-positive Jurkat cells were quantified by flow cytometry 72 h after infection at a MOI of 5E2 and 1E3 (data are shown as mean ± SD of six distinct samples (n=6)). b) Evaluation of the transduction efficiency of rAAV2 and rAAV2-Lys-CD45 in presence of 500 µg/mL of heparin in HeLaT and Jurkat cells. Both rAAV2 vectors were carrying an eGFP reporter gene expression cassette under the control of a ubiquitous promoter. GFP-positive Jurkat and HeLaT cells were quantified by flow cytometry 48 h after infection at a MOI of 5E3 in presence of 0 µg/mL or 500 µg/mL of heparin. The results are expressed as "% residual transduction" which corresponds to the ratio between transduction in presence and in absence of heparin (data are shown as mean ± SD of six distinct samples (n=6)). Statistical significance was assessed using Mann-Whitney test (*p*<0.05; **p*<0.01).</figcaption></figure>

Beyond its impact on rAAV2 tropism, the effect of nanobodies grafting on vector immunogenicity was investigated. To this end, the recognition of rAAV2 and rAAV2-Lys-CD45 by human antibodies was evaluated using anti-AAV2 IgG seropositive human serum in an ELISA assay. The results, expressed as IgG titers, (i.e., the last dilution of human serum at which anti-AAV2 IgG were detected; Fig. S21), revealed lower recognition of rAAV2-Lys-CD45 by human anti-AAV2 IgG compared to rAAV2, with titers of 1/1260 and 1/2650, respectively. Interestingly, this suggests that protein grafting could be a promising strategy to enhance the tissue specificity of rAAV while reducing its recognition by the immune system.

To complement this study, azido conjugations and post-functionalizations were successfully conducted on rAAV8 (Fig. S22). The results demonstrate higher azido-conjugation on rAAV8 tyrosine residues compared to lysine residues, as well as the successful post-functionalization of rAAV8-Tyr-N₃ and rAAV8-Lys-N₃ with the fluorescein moiety and the anti-CD62L nanobody, respectively. Given the significant homology among AAV serotypes (except for AAV5)⁵¹, the successful modifications of rAAV2 and rAAV8 underscore the versatility of this strategy and highlight the potential of chemical bioconjugation for viral capsid engineering.
Ultimately, additional proteins with relevant specific targeting properties will need to be conjugated to the viral capsid to explore the range of cell-specific vectors, their promising tissue-targeted tropism, and their potential to evade the immune system. We hope this work will contribute to reducing the toxicity of gene therapy and decreasing the doses administered to patients.

## CONCLUSION

We propose here a new, simple and rapid technological platform for the custom grafting of small molecules or proteins onto rAAV vectors, to improve their efficacy and specificity. To this end, we demonstrated a capsid engineering sequence initiated by chemical capsid labelling with bioorthogonal groups (azido, DBCO) and followed by an optimized SPAAC reaction. This direct and rapid sequence enabled access to rAAV-proteins conjugates from natural serotypes 2 and 8, avoiding time-consuming studies on insertion sites and production conditions (otherwise needed if proteins are genetically incorporated in the rAAV sequence). Modulated levels of functionalization can be achieved by using different bioconjugation reactions or by targeting different amino acid residues, allowing to adapt the strategy in cases of aggregation or toxicity issues with the labelling moiety. The biological interest of rAAV-protein conjugates will be further investigated in forthcoming work. Nevertheless, this study already demonstrates that rAAV-nanobody conjugates can transduce targeted cells in presence of a rAAV binding inhibitor, which is promising for the development of highly cell-specific vectors. We expect that this bioorthogonal chemical modification of the AAV capsid will represent an easy and versatile strategy to unlock numerous challenges faced in the field of gene therapy. We also believe that this strategy may open up new applications for chemically modified capsids, using, for example, *in vivo* SPAAC reactions which have already been described in the literature with other models⁵².

## ACKNOWLEDGMENT

The authors thank the vector core of TaRGeT, UMR 1089 (ViVeM, INSERM and Nantes Université, which is a bioproduction and biotherapy national integrator (ANR-22-AIBB-0001), http://umr1089.univ-nantes.fr) for the production of the rAAV vectors used in this study. This research was supported by the Fondation d'Entreprise Thérapie Génique en Pays de Loire, the Centre Hospitalier Universitaire (CHU) of Nantes, the Institut National de la Santé et de la Recherche Médicale (INSERM), Nantes Université, and by grants from the French National Agency for Research ChemAAV (ANR-19-CE18-0001)). The authors acknowledge the Cytocell - Flow Cytometry and FACS core facility (SFR Bonamy, BioCore, Inserm UMS 016, CNRS UAR 3556, Nantes, France) for its technical expertise and help, member of the Scientific Interest Group (GIS) Biogenouest and the Labex IGO program supported by the French National Research Agency (n°ANR-11-LABX-0016-01).
**Credit authorship contribution statement**

Maia Marchand (Conceptualization; Methodology; Writing – original draft; Writing – review & editing), Mohammed Bouzelha (Conceptualization; Methodology; Validation), Sébastien Depienne (Conceptualization; Methodology; Visualization; Writing – original draft), Karine Pavageau (Methodology; Validation), Roxanne Peumery (Methodology), Denis Loquet (Methodology), Dimitri Alvarez-Dorta (Methodology; Validation), Mickaël Guilbaud (Methodology; Validation), Mikaël Croyal (Methodology; Validation), Aurélien Dupont (Methodology; Validation), Oumeya Adjali (Funding acquisition; Project administration; Resources; Supervision), Sébastien G. Gouin (Supervision; Writing – original draft), David Deniaud (Conceptualization; Funding acquisition; Methodology; Resources; Validation; Writing – original draft; Writing – review & editing), Mathieu Mével (Funding acquisition; Supervision; Validation; Writing – original draft; Writing – review & editing).

**Conflicts of interest**

M.M., D.D., S.G.G. and S.D. are inventors on patents including the technology described in this manuscript. No potential conflicts of interest were disclosed.

**Data availability**

All relevant data are available from the authors on request and are included within the manuscript.

**Appendix A. Supplementary data**

Experimental procedures, chemical synthesis, ¹H NMR, ¹³C NMR, supplementary figures and gating strategies.

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