Artificial viral vectors constructed from bacteriophage T4 show promise for advanced gene delivery

By Bhavana KunkalikarReviewed by Benedette Cuffari, M.Sc.Jun 1 2023

In a recent study published in the journal Nature Communications, researchers present a method for constructing artificial viral vectors (AVVs) using the established structural components of bacteriophage T4.

Study: Design of bacteriophage T4-based artificial viral vectors for human genome remodeling. Image Credit: Kjpargeter / Shutterstock.com

What are viral vectors?

AAVs and lentiviruses (LVs) have been modified to transport therapeutic deoxyribonucleic acid (DNA) and ribonucleic acid (RNA); however, viral vectors are associated with certain limitations. For example, the delivery of therapeutic genes is limited, and it is challenging to include other necessary therapeutic molecules for complex repairs.

T4 is a highly efficient virus with an almost 100% infection rate and a rapid replication cycle of approximately 20-30 minutes/cycle. These characteristics indicate that T4 is an excellent foundation for constructing AVVs.

Assembly of T4 artificial viral vectors

A virus structure mimics known as T4-AVVs was created by assembling purified biomaterials in a sequential manner. A pentameric packaging motor was constructed on the portal vertex of an empty capsid shell that was purified from E. coli infected with the T4 phage mutant. This was achieved by the addition of the motor protein gp17 to the reaction mixture.

Foreign DNA is introduced into the capsid interior through the addition of linearized plasmid DNA and adenosine triphosphate (ATP) to the assembly reaction. The T4 packaging motor moves DNA into a capsid in a processive manner by capturing and translocating the bacteriophage from one end to the other. Successive packaging of DNA molecules can occur multiple times, thereby leading to a full head.

RNA, proteins, and their complexes are attached to packaged head particles through highly antigenic outer capsid (Hoc) and small outer capsid (Soc) protein interactions.

The researchers also coated particles with cationic lipid molecules, as they hypothesize that cationic lipids would spontaneously accumulate on the T4 capsid through electrostatic interactions due to the high density of negative charges in the T4 capsid. T4-AVV nanoparticles have the same structure as naturally enveloped viruses, including a capsid shell, lipid coat, and packaged "genome."

Lipid-coated T4-AVVs are highly effective in delivering genetic payloads into human cells. Furthermore, when co-packaged with distinct linear plasmids, these AVVs efficiently transduced reporter plasmids into HEK293T cells.

On average, each nanoparticle contained approximately five molecules of luciferase plasmid (Luci) and green fluorescent protein (GFP) reporter plasmid. GFP fluorescence was used to determine the transduction efficiency, which was about 100%.

T4-AVV transduction was compared to the commonly used AAV2, both of which contained the same sequence plasmid. However, T4-AVVs had four to 19 times higher luciferase expression than AAV2, which could be attributed to the ability of T4 to package around eight Luci plasmid molecules in each head, thereby allowing for the delivery of several copies of the genetic payload in a single transduction. In contrast, AAV2 is restricted to delivering one copy at a time.

T4-AVVs are unique due to their large capacity head that can hold up to approximately 171 Kbp. This allows for the delivery of large therapeutic genes, including the full-length coding DNA of the 11 Kbp human dystrophin gene, which cannot be achieved by current LV and AAV vectors.

Genome editing AVVs

Different genome editing AVVs were created by combining all editing molecules into a single AVV in various arrangements. The first set of AVVs contained plasmids with expressible Cas9 and gRNA genes, in which the Cas9 N-terminus was fused with the nuclear localization sequence (NLS) PKKKRKV71 and codon-optimized to create NLS-Cas9. Subsequently, Cas9 was transported into the nucleus to perform genome editing, whereas gRNA was directed towards the AAVS1 safe harbor locus, or the PPP1R12C locus, on chromosome 19 from the human genome.

T4-AVVs were used to target the hemoglobin beta gene (HBB) on chromosome 11 of the human genome to perform genome editing at a therapeutically significant location. AVVs assembled with Cas9-HBB gRNA ribonucleoprotein (RNP) complexes (AVV4) achieved approximately 20-25% editing at the targeted site.

Furthermore, AVVs were created by exhibiting two gRNA-RNP complexes on a single AVV (AVV5), with one complex aimed at the HBB site and the other at the AAVS1 site. Multiplex AVVs successfully edited the genome at two different chromosome sites, with approximately 20% at the HBB site and almost 30% at the AAVS1 site.

Conclusions

The researchers developed a new category of nanomaterial known as a bacteriophage-based artificial viral platform, which can be produced in a test tube through an assembly-line approach. AVVs have a structure similar to natural viruses; however, further research is needed to understand how they enter cells, escape endosomes, disassemble, and move within cells to improve their delivery.

This novel platform is ready for therapeutic use and can correct defects within primary human cells, both inside and outside the body. Developing lipid nanoparticle technologies and using traditional and clustered regularly interspaced short palindromic repeats (CRISPR) engineering techniques would be necessary to create specific payload designs.

Notably, some safety concerns may arise during the transition of T4-AVVs to the clinic, including potential unwanted reactions by the host immune system or off-target influences. Therefore, the T4-AVV platform is currently being investigated for its safety and effectiveness.