Background

There has been a desperate need for new and efficient delivery vehicles that can transport large cargos of genes and proteins into human cells to stimulate the production of therapeutic biomolecules and/or to repair cellular and genetic defects. Such vehicles will allow translation of the rapidly emerging technologies such as CRISPR, CAR T cells and so on, into therapies, both for mass applications as well as for personalized medicine (Stewart et al., 2016).

Assembling nanoparticles with different properties into a hybrid complex is a powerful strategy for developing novel functional materials, as these hybrid complexes exhibit collective and collaborative attributes, some of which might be different from those exhibited by the individual particles (Ghosh et al., 2012; Wang et al., 2014). Virus nanoparticles representing a fascinating class of protein-polynucleotide supramolecular structures are an ideal natural resource to construct such hybrids. Considering different shapes and sizes ranging from tens to hundreds of nanometers and their documented ability for intracellular delivery, combining different viral building blocks into a multifunctional nanoparticle will greatly accelerate future therapies.

Molecular and genetic analyses led to the engineering of an empty phage T4 shell (head or capsid) lacking the outer capsid proteins Soc (small outer capsid protein; 870 copies/head; 9 kDa) and Hoc (highly antigenic outer capsid protein; 155 copies/head; 41 kDa), neck, or tail as a delivery vehicle (Zhang et al., 2011). Both Soc and Hoc are non-essential for phage infection and exhibit high binding affinity and specificity to T4 capsid surface. Soc binds as a trimer at the quasi three-fold axes whereas Hoc binds as a monomer at the center of each gp23 capsomer (hexamer). Foreign proteins can be efficiently displayed on T4 head through fusion to N and C termini of Soc or the N terminus of Hoc which are well exposed (Tao et al., 2013).

The 25 nm adeno-associated virus (AAV) has been extensively used as a gene delivery vehicle but it has a very small capacity and cannot deliver proteins. The 120 x 86 nm prokaryotic virus, the bacteriophage T4, on the other hand has a large capacity but it lacks natural mechanisms to enter human cells. It has no tropism to human cells, has no known toxicity or pathogenicity, and exhibits no preexisting immunity. We reported the development of the phage-virus hybrid vector by integrating these two most well-characterized delivery vehicles into one (Figure 1) (Zhu et al., 2019). A specific biomolecular avidin-biotin interaction was selected as a driving force to mediate the interaction between the T4 head and AAV particle. It is also one of the strongest no-covalent interactions known in nature (Kd = 10^-15 M), making it ideal as a molecular bridge between the two nanoparticles (Jain and Cheng, 2017). We engineered it in such a way that all the different compartments of the T4-AAV nanoparticle could be utilized for loading cargo molecules. These include the interior of the T4 capsid shell that can accommodate 170 kbp of DNA cargo (Zhang et al., 2011), the interiors of attached AAV shells each accommodating 5 kbp of DNA cargo (Muzyczka, 2014), and the exterior of the T4 shell that can be decorated with up to 1,025 molecules of protein cargo (Li et al., 2007; Shivachandra et al., 2007; Tao et al., 2013 and 2018). This system has been optimized such that varieties of customized cargos can be prepared by simple mixing of interacting components that have nanomolar or sub-nanomolar affinities and by using a powerful DNA packaging motor (Leffers and Rao, 1996; Fuller et al., 2007). Furthermore, different cargo molecules can be mixed and matched, quantified, and tuned as desired.


Figure 1. Schematic of DNA & protein carried T4 and T4-AAV hybrid vector

In this protocol, we describe the construction, expression, purification, and biotinylation of Soc and BAP-Hoc (BAP, Biotin Acceptor Peptide), and the assembly of Soc-biotin-avidin and Hoc-BAP-biotin-avidin. Soc and BAP-Hoc were biotinylated in vitro using biotin N-Hydroxysuccinimide (NHS) esters and BirA ligase, respectively. NHS-activated biotins react efficiently with primary amino groups (-NH2) of Soc protein (the side chain of lysine residues and the N-terminus) to form stable amide bonds. BirA biotin ligase site-specifically biotinylates a lysine side chain within a 15-amino acid BAP peptide fused to the N terminus of Hoc protein. We also provide a description of the production, purification, and modification of AAV and T4 head. Finally, we describe the methodology for generating the hybrid T4-AAV particle using biotinylated AAV, T4 head, and the “linker” molecules, Soc-biotin-avidin and Hoc-BAP-biotin-avidin (Figure 2). This protocol describes the assembly of hybrid T4-AAV vector in ~2 weeks. Although this study focused on T4 and AAV to conceptually demonstrate the utility of combining distinct viral building blocks, in a broader context, other hybrid vectors containing the T4 ‘carrier’ complexed with eukaryotic viruses, bacteria, or polymers using bioaffinity attachment technology can be envisioned for delivery of multivalent gene and protein therapeutics (Akin et al., 2007; Yoo et al., 2011).


Figure 2. Workflow for construction of T4-Soc-AAV (A) and T4-Hoc-AAV (B) vectors. The T4 capsid lattice is decorated with two nonessential outer capsid proteins, Soc and Hoc. Soc and BAP-Hoc proteins were expressed in E.coli, purified by chromatography, and biotinylated. Two bridge molecules were constructed to attach AAV to the T4 head, Soc-biotin-avidin and Hoc-biotin acceptor peptide (BAP)-biotin-avidin. Soc and Hoc attach to the T4 head, and avidin attaches to biotinylated AAV (bioAAV) on the other side. T4-Soc-AAV (A) and T4-Hoc-AAV (B) conjugates were then purified by Ni²⁺-NTA agarose chromatography.