Developing a Targeted AAV System Using GFP Nanobody

Gene therapy is game-changing technology in the realm of medicine as it represents a revolutionary approach to treating inherited diseases at their root cause – the genetic level. By introducing, modifying, or correcting genes within a patient's cells, gene therapy aims to address genetic disorders, but it may be also helpful to fight certain cancers and other conditions that arise from faulty or missing genetic information. Gene delivery systems play a pivotal role in this process by facilitating the introduction of therapeutic genes into target cells. These delivery systems can be viral (e.g., adeno-associated virus, lentivirus) or non-viral (e.g., liposomes, nanoparticles). The ideal delivery system should allow targeted, precise, and effective treatments for a wide range of medical conditions.

An Adeno-associated virus (AAV) is a small, non-enveloped, single-stranded DNA virus that has earned significant attention in the field of gene therapy. AAV is considered a promising vector for gene delivery due to its safety profile, low immunogenicity, and ability to infect both dividing and non-dividing cells. AAV vectors are now commonly used to transport therapeutic genes into target cells for gene therapy applications. Their small size limits their capacity for exogenous genetic material, but their ability to integrate into the host cell genome ensures durable transgene expression. AAV vectors can be pseudotyped with capsid proteins from different serotypes to target specific tissues, making them a valuable component in advancing gene therapy approaches.

Our objective was to develop an AAV targeting system based on a nanobody display approach. As a proof-of-principle, we utilized a GFP nanobody to create a recombinant AAV-capsid capable of specifically targeting cells with GFP displayed on their surface. The variable and surface exposed VR-4 and VR-8 regions of the AAV2 capsid protein were utilized to generate in-frame fusions with the GFP nanobody. We designed multiple linker sequences to ensure optimal positioning of the nanobody for effective antigen-binding paratope functionality. During this practical course, the AAV2 capsid was modified to allow seemless integration of the GFP nanobody. Furthermore, several linker-nanobody configurations were designed and explored for integration into the capsid sequence. Consistent with findings from other groups, the modification of the AAV capsid shows promise of high-specificity gene delivery tools for precise applications in research and healthcare.

Gene Therapy

Gene therapy is a cutting-edge medical approach aimed at treating or preventing diseases by altering the genetic material within a person's cells. It involves the introduction, removal, or modification of specific genes to correct genetic mutations, replace faulty genes, or regulate their activity (Wagner et al., 2021). Currently there are gene therapy treatments in the market such as Zolgensma, a novel gene therapy product designed to treat spinal muscular atrophy using non-replicating, self-complementary AAV serotype 9 (AAV9) vector containing the human survival motor neuron gene (SMN1) constructed to be episomal within the host cells and provide continuous expression of the therapeutic protein (Michels et al., 2021), or nanobodies that are used in a “Lego brick like fashion” that was approved for clinical use in 2019 for the first time, for the treatment of acquired thrombotic thrombocytopenic purpura (Hambach et al., 2022). This field holds great promise for a wide range of medical conditions, including inherited genetic disorders, certain types of cancer, and various other diseases. While still an evolving field with ongoing research and clinical trials, gene therapy represents a potential revolution in healthcare, offering the prospect of targeted and personalized treatments for previously incurable or difficult-to-treat conditions (Kotterman and Schaffer, 2014; Michels et al., 2021).

AAV as Gene Therapy Vector

The challenge of effective gene therapy is the identification of suitable vectors for efficient gene transfer into the target tissues. A major benefit of AAV is its ability to integrate the genetic load into the host cell genome. This ability ensures long-term and stable expression of therapeutic genes within the host genome translating into sustained therapeutic benefits over an extended period. The reduced risk of immune responses associated with integrating strategies, as compared to non-integrating approaches, enhances the safety profile of AAV-mediated gene delivery. Additionally, integration allows for the attainment of therapeutic effects with lower vector doses, minimizing the potential for adverse reactions and promoting overall safety. Therefore, one focus of further developing AAV as a gene therapy vector is on improving the effectiveness of integration strategies, especially in addressing genetic disorders that require continuous and stable expression of therapeutic genes (Pupo et al., 2022).

The other major research focus in the context of AAV vectors is improved cell type specific targeting of AAV. These non-replicative, non-enveloped, small ssDNA viruses are known to be a safe and effective gene delivery vector. However, some AAV serotypes show a wide tropism for human cells, which limits the ability to deliver the gene of interest into a specific target cell type (Herrmann and Grimm, 2018).These obstacles are tried to be overcome by manipulating the capsid proteins in the form of mutations, peptide or ligand insertions that would reduce broad binding to a wide variety of human cells and rather increase the selectivity for specific target cell types or allow binding to murine cells (Hambach et al., 2022).

Optimizing Delivery Systems

AAV capsids are essential components that encapsulate the viral genome and are crucial for the virus's ability to infect cells. By modifying the capsid protein, the cell tropism of the virus can be altered. One approach entails chimeric AAV capsids, which are engineered viral protein shells composed of elements from different naturally occurring AAV serotypes. Researchers create chimeric AAV capsids by combining specific regions or domains from different AAV serotypes. This process aims to leverage favorable characteristics from various serotypes, such as improved transduction efficiency, altered tissue tropism, reduced immunogenicity, and other features beneficial for gene therapy applications (Pupo et al., 2022). A second approach uses the insertion of well-characterized binding domains into the capsid framework. For example, DARPINs and nanobodies have been used as targeting devices for AAV due to their advantageous properties such as their high solubility and flexibility. For example, the AAV2 VP1 capsid protein has been successfully modified with a CD38-targeting nanobody allowing increased transduction of CD38-expressing cells (Eichhoff et al., 2019).

Aim

Targeted AAV System Development: The project specifically focused on creating a simple targeted AAV system using a nanobody display strategy. It sought to integrate a GFP nanobody onto the AAV2 capsid protein, testing the efficiency and action mode of this system. The project also involved experimenting with different linker sequences for efficient antigen-binding.

Generation of Insertion Sites on Capsid Fragment

Engineering AAV Capsids: Two potential genomic locations in the AAV capsid protein were identified for modification. These variable surface regions (VR IV and VR VIII) were chosen due to their variability across different AAV serotypes, allowing for structural modifications without affecting the protein's stability.

Amplification of Nanobody Sequence with Linker Combinations

Primer Design for Linker Sequences: The project involved designing and producing various GFP nanobody-linker combinations. These linkers, particularly Glycine-Serine (Gly-Ser) linkers, were used to create functional, stable recombinant fusions of a nanobody with the viral capsid.

The samples from the first Nested PCR step for GFP-nanobody-linker fragment amplification initially showed much larger (above 500 bp) or no fragments, before we identified the problem as wrong reverse primers. (A) refers to EGKS-Q/GGGS linker combination whereas (B) refers (GGGGS)x3/GGGS combination. Positive Control (+) refers to the sample produced with alternative, known primers for the same GFP-nanobody sequence which shows the correct band size with low size difference to our GFP-nanobody linker design as 40 bp.

Cartoon model of prospective fusion constructs with the GFP nanobody and AAV capsid protein. AAV residues of VR-4 region are coloured in red whereas green for VR-8 region. GFP Nanobody NMR Structure is taken from Mueller, G.A. AAV capsid protein image is taken from the cryo-EM Structure of genome containing AAV2 deposited to PDB by Bennett, A.D., Mckenna, R. Scaling of the nanobody and the capsid protein was assumed to be 1:5.7 as an AAV is a virus in 26 nm diameter and the height of a GFP nanobody is around 4.8 nm (Muyldermans, 2013; Rayaprolu et al., 2013).

We preferred short Glycine-Serine linker and on the C terminus as it is located on the far side of the GFP binding paratope. We also included a longer design for C terminus with the peptide sequence KESGSVSSEQLAQFRSLD which is shown to be effective for construction of a bioactive scFv (Chen et al., 2013), as a flexible linker alternative. For the N terminus, we have either EGKSSGSGSESKSTQ or (GGGGS)x3 sequences as linker to create the desired positioning of the nanobody on top of the surface of AAV capsid. Finally, we have included restriction sites of AgeI for fusion and HindIII located at the C-terminus for determining the orientation of the GFP nanobody. To achieve the nanobody-linker sequence we used two primers per long linker sequences in a nested PCR.

The insertion of a GFP nanobody onto an AAV capsid protein for gene delivery can introduce potential challenges that could impede the efficacy of transduction that needs to be investigated closely. Issues such as steric hindrance may develop due to the mass created by the GFP nanobody on the AAV capsid, affecting the proper interaction of the capsid with cellular receptors and ultimately interfere with the viral entry into target cells (Becker et al., 2022). Insertion of the GFP nanobody may alter the natural functionality of the capsid, potentially affecting its binding to target cells and thus the transduction process. Further, the introduction of a foreign protein might increase the immunogenicity of the vector, triggering host immune responses that could clear viral particles and hinder gene delivery (Ertl, 2022). The stability of the modified capsid and its production may be compromised, leading to premature degradation for instance. Ensuring proper display, preserving natural tropism, maintaining payload capacity, and addressing potential alterations in intracellular trafficking are essential considerations to mitigate these challenges and optimize the effectiveness of the AAV vector in gene delivery applications depending on the target cells (Cabanes-Creus et al., 2020).

Another research group achieved promising results of high specificity using a very similar AAV2 capsid protein design, with the nanobody positioned at VR-4 (GH3-GH4 loop) in their gene delivery experiments in mouse splenocytes (Michels et al., 2021). Despite falling short of our final objective, this assay showed the necessity to improve our design strategies and investigate further, consistent with findings from other groups that indicate the potential of a such high specificity gene delivery tools in research and healthcare.

A schematic representation of the production process of the GFP nanobody displaying AAV with red-fluorescence gene package. Recombinant pAAV vector contains a reporter gene such as a different fluorescent protein gene as mCherry. AAV-293 cells function as the host cells for AAV vector production. pHelper plasmid carries genes derived from adenovirus that provide essential functions for the replication of AAV in the presence of adenovirus helper functions. AAV-293 cells are engineered to express the adenovirus genes, making them permissive for AAV replication. pAAV-VR4-GFPnb or pAAV-VR8-GFPnb plasmids carry AAV-2 replication (Rep) and GFP-nanobody displaying capsid (Cap) genes (depending on the location of the insertion named as VR-4 or VR-8), providing the necessary factors for AAV replication and capsid protein synthesis. The recombinant expression plasmid, pHelper, and pAAV-RC are co-transfected into the AAV-293 cells. This co-transfection supplies all the trans-acting factors required for AAV replication and packaging. The co-transfected AAV-293 cells undergo a series of steps, including AAV replication, packaging of the viral genome, and assembly of viral particles. Recombinant AAV-2 viral particles are harvested from the AAV-293 cells. The viral particles can be collected from the cell culture medium or the cells themselves. The cell lines and plasmids are named from Agilent Technologies’ AAV Helper-Free System.

To facilitate the identification and quantification of the specificity of the construct, we would involve integrating a reporter gene such as a different fluorescent protein gene as mCherry as a recombinant pAAV vector. The commercial AAV Helper-Free System by Agilent Technologies can be used for producing viral packages. The designed construct would then be subjected to cellular transduction targeting GFP-displaying mammalian or murine cells. In addition to evaluating specificity via the expression of red fluorescence, assessments would include measuring the transduction efficiency to prove that the addition of nanobody onto capsid protein would not interfere with the efficiency with flow cytometry, western blotting and more. Positive GFP-nanobody-AAV capsid variants would be used for in vivo studies for physiological evaluation, focusing on tissue-specific targeting and biodistribution. The optimized GFP-nanobody-AAV capsid construct would serve as a starting point of a promising tool for precise gene delivery.

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