Venigalla B. Rao, PhD, has been studying bacteriophages for more than four decades, mostly in small laboratories with limited personnel and funding. But having a mere eight-person lab (four post-docs, two graduate students, and two research technicians) hasn’t deterred Rao from pursuing his goal of using phages to transform gene therapies and personalized medicine.

In a new study published in Nature Communications, Rao and his team show how to construct artificial viral vectors (AVVs) using an assembly-line technique that allows for the delivery of large biomolecular payloads, such as 171 kilobases of DNA, thousands of protein molecules, and RNAs. In order to edit the human genome—recombine or replace genes, alter or silence gene expression—they have redesigned the well-characterized structural components of bacteriophage T4 to accomplish these tasks.

In order to program and deliver therapeutic biomolecules, Rao and colleagues used T4 bacteriophages to build AVVs with a large internal volume and a large external surface. Proof-of-concept experiments showed the viability of these AVVs for use in genome engineering by loading them with protein and nucleic acid cargo. The full-length dystrophin gene and other molecular operations to modify the human genome were successfully carried out in the lab using the platform. The AVVs have a high yield and low cost of production, and the nanomaterials have been shown to be stable for several months.

Rao hopes that this technique could eventually be used to treat a variety of common and rare disorders in humans, although more study is needed to determine its safety.

Venigalla Rao Lab
The Rao Lab. From left to right: Wenzheng Guo, Xiaorong Wu, Rao, and Jingen Zhu. [Patrick G. Ryan]

The phage whisperer

After finishing his dissertation on enzyme engineering at the Indian Institute of Science, Rao began his postdoctoral work on phages in the lab of the late Lindsay W. Black at the University of Maryland School of Medicine. As Rao explained to GEN, “when we started this, very little was known about DNA packaging—it was a big, mysterious black box.”

In 1989, Rao joined the faculty of the Catholic University of America in Washington D.C., where he established his own research program. At times Rao admits he struggled because it’s very basic research, and phages are not the most popular organisms to work with.

“Neither NSF nor NIH were really interested in funding phage research that enthusiastically,” said Rao. “But I always believed in phages because these are good model systems to really understand the basic mechanisms of DNA packaging, there’s a lot we can manipulate genetically, and they are inexpensive. I can do it with students and a small research lab with Petri dishes and LB media.”

Rao persisted, and his efforts were ultimately funded by the National Science Foundation. Then, in 1999, Purdue University’s Michael Rossmann, PhD, who had become famous for his X-ray crystallographic analysis of the common cold virus, heard about some intriguing packaging mutants being developed in Rao’s lab. Working together, Rossman and Rao became a driving force in deciphering the components of bacteriophage DNA packaging.

Taming T4

Around this time, Rao realized the importance of applying this knowledge in the field of medicine. Because of the rising interest in the development of biodefense vaccines following the anthrax scare in the United States beginning on September 18, 2001, one week after the September 11 terrorist attacks, Rao initially focused on vaccines and how to display pathogen antigen molecules on the phage capsid surface, which received decent funding.

In parallel, Rao kept digging into the inner workings of phage packaging, noting that the capsid nanoshell of phages could be engineered for purposes beyond vaccines. “I felt gene therapy is where it might make the biggest impact because the gene therapy field has been struggling because of the limited capacity and engineering flexibility of the vectors available,” said Rao.

Rao continued his work on using phages as a vehicle for gene therapy in human cells, as published in a PNAS article with his colleagues in 2013. Rao claims that the technology has been nearly optimized in his lab to the point where many things are possible with that nanoshell. “We can package multiple genes and plasmids in the same capsid and display molecules to target the nanoshells to specific cells and tissues,” said Rao.

As human pathogens have lipid bilayers (unlike phages), Rao reasoned that the nanoparticles needed to be coated in lipids in order to translocate into human cells more effectively. He was surprised to discover that the surface of T4 phages was naturally receptive to lipid coating. After experimenting with various conditions, he was able to achieve this.

Although phages are simple to work with genetically, Rao claims that it is still a tedious process to generate mutants. In order to quickly generate any mutants they required, Rao’s team developed CRISPR engineering technology and identified a mutant nanoshell that resulted in five times the delivery efficiency of the wild-type capsid.

In his team’s latest work, Rao demonstrates how they have optimized this technology in the Nature Communications report, demonstrating their ability to package massive amounts of DNA, RNA, and proteins, as well as complexes combining these biomolecules, such as CRISPR systems with guide RNAs.

While Rao’s team has demonstrated the feasibility of their system in immortalized cell lines like 293 T cells, they are currently working to implement it in primary human cells and human embryonic stem cells, and even in an in vivo animal model. The goal, according to Rao, is to get the system into a mouse model as soon as possible so that it can be brought to the clinic in the not-too-distant future.

Previous work from Rao’s lab, which was posted on bioRxiv about a year ago, demonstrated cell-specificity with the T4 phage system, targeting CD4+ primary human T-cells.

bacteriophage T4 capsid nanoshell
Structural features of the 120 x 86 nm bacteriophage T4 capsid nanoshell at near-atomic resolution (Left—front view; Right —cross-section view showing empty interior). The capsid subunits are shown in different colors: hexameric major capsid protein gp23* (cyan), pentameric vertex protein gp24* (magenta), dodecameric portal protein (red), trimeric small outer capsid protein (Soc; orange), and monomeric highly antigenic outer capsid protein (Hoc; yellow). [Venigalla B. Rao; Victor Padilla-Sanchez, Andrei Fokine, Jingen Zhu, and Qianglin Fang]

From concept to clinic

For Rao, however, the available resources (money and facilities) are still the bottleneck. “My research lab is really small—all this took like seven plus years to develop this technology using a large amount of basic information,” said Rao. “We really need funding to build the infrastructure needed to take it to the clinic as quickly as possible, especially that the adeno-associated viruses (AAVs) and lentiviruses are already in the clinic. The biotechnology industry is more interested in investing in something that’s already in the clinic. Hopefully, the paper will show a level of enthusiasm to maybe take some risks here. The pay-off could be tremendous.”

Indeed, this has not been done clinically with T4 phages. But Rao is certain that since T4 bacteriophages are a natural resident of the gut microbiome, and based on the data from the vaccine projects, they can be very safe for biomedical applications. “As part of our vaccine projects, we have done numerous intramuscular and intravenous immunizations in a variety of animals, including rhesus macaques, and they all turned out to be quite innocuous,” said Rao. “There was no incident of any serious side effect of administering fairly large quantities of T4 bacteriophages.”

At Catholic University, Rao said his lab actually designed COVID vaccines, including a nasal vaccine, which he is trying to bring to global market. Rao has ideas to develop an efficient GMP process to manufacture T4 phages for biomedical applications and said that, as far as he can tell, it should be much simpler than making AAVs and lentiviruses because T4 phages are produced in E. coli and don’t require as many safety considerations (although immunogenicity might be an issue).

Rao is confident that, unlike traditional viral vectors, T4 phages have impressive flexibility and engineering capacity. While he expects some challenges, he believes that this work could really open up a new space for gene therapy beyond monogenic diseases.

“The T4-AVVs could be used with more complex diseases and for personalized medicines because we can more easily, using the same system, slightly change the biomolecule to personalize it depending on the mutation in the genetic disease,” said Rao. “I think it would be much more rapid to actually make those changes and individualize it. We are highly inspired and excited about it. Hopefully, we can accelerate the process and make it happen.”

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