Our project aims to construct a novel defense system against avian influenza viruses that currently threaten global food security, utilizing synthetic biology as our primary approach.

Rather than developing orthodox defense systems such as drugs or vaccines that inhabit viral entry into cells or viral replication, we are developing an innovative mechanism that prevents viral amplification within animal hosts by "actively inducing apoptosis in cells infected with influenza virus." Our ultimate goal is to suppress highly pathogenic avian influenza(HPAI) pandemics, thereby making significant contributions to improving food security issues and maintaining public health.

Chickens constitute an indispensable poultry species in global food production systems. Annually, over 100 million tons of chicken meat and more than one trillion eggs are produced and consumed worldwide. Compared to other livestock such as swine and cattle, chickens represent a more environmentally sustainable and economically efficient protein source. Indeed, the feed conversion ratio required to produce a given quantity of meat is approximately one-fifth that of beef production [1]. Due to these advantages, demand has been steadily increasing in recent years, with chicken meat and eggs currently accounting for approximately 12% of global dietary protein intake [2].

However, recent highly pathogenic avian influenza (HPAI) outbreaks have caused recurrent and severe damage to poultry operations across multiple regions. Between 2005 and 2024, approximately 633 million chickens are estimated to have succumbed to highly pathogenic avian influenza globally [3].

Under current HPAI biosecurity guidelines, all chickens in an infected poultry house, as well as those in epidemiologically linked houses, must be culled. While this measure is essential for preventing the spread of the virus, the sheer number of lost birds results in enormous economic damage. In fact, massive funds are being spent by various governments to compensate poultry farmers affected by HPAI. For instance, the US government's expenditure for countermeasures in 2024 exceeded \$1.4 billion, with \$1.25 billion of that total allocated to compensation and indemnification payments [4]. In this manner, HPAI is currently threatening global food security on a massive scale.

Highly pathogenic avian influenza virus (HPAIV) constitutes a subtype of influenza A virus. Influenza A viruses demonstrate broad host tropism, infecting numerous animal species including not only humans and domestic poultry such as chickens, but also equines, swine, canines, pinnipeds, and cetaceans.

Influenza A viruses are single-stranded RNA viruses. They are classified according to their surface antigens, particularly hemagglutinin (HA) and neuraminidase (NA).

Representative HPAIV subtypes that have demonstrated significant pathogenicity in recent years include H5N1 and H7N9.

Given that influenza A represents a zoonotic pathogen, instances of HPAI transmission to humans have been documented. As of 2019, human infections with the H5N1 Avian Influenza virus, primarily transmitted from poultry such as chickens, had been confirmed in various countries worldwide. The total number of confirmed human cases reached 860, of which 454 people died [5]. Such cross-species influenza viruses possess the potential to precipitate large-scale pandemics, as exemplified by the 1918 Spanish influenza pandemic.

Consequently, HPAI represents a pathogen requiring mandatory intervention strategies. However, current vaccination-based viral prophylaxis remains inadequate, with poultry vaccination typically failing to prevent clinical manifestation in most cases. This therapeutic limitation stems from the inherent high virulence characteristics of HPAI.

HPAIV demonstrates the capacity to cause mortality in chickens with near certainty within 48 hours following invasion, even at minimal viral loads (100-1,000 viral particles). Conventional low-pathogenicity influenza viruses can replicate only in specific tissues such as pulmonary epithelium cells.

However, HPAIV possesses unique characteristics that enable infection and replication across multiple organ systems throughout the avian host [6].

Consequently, even minimal initial viral inoculum can undergo extensive amplification and dissemination systemically, resulting in severe pathogenic manifestations.

The exceptionally rapid emergence of viral variants further complicates vaccine development strategies.

Given these circumstances, there is an increasing demand for innovative antiviral defense strategies capable of attenuating the potent virulence of HPAIV and preventing clinical manifestation.

We decided to counter HPAI, a potent viral pathogen, through a synthetic biology-inspired approach. A fundamental principle in synthetic biology involves transplanting beneficial systems from one organism to another.

Our attention was drawn to the phage infection defense mechanisms possessed by E. coli.

The evolutionary arms race between bacteria and phages has been extensively documented in recent years, with numerous well-known examples such as Cas9. During the initial phase of our research activities, we developed an interest in the PARIS system [7], an E. coli phage infection defense mechanism, and conducted investigations in this field.

A common feature among these phage defense systems is that E. coli infected by phages undergo programmed cessation of cellular activity, thereby preventing phage replication within the cell and subsequent dissemination to neighboring bacterial cells. We conceived the possibility of applying this E. coli-phage relationship paradigm to the interaction between avian cells and HPAIV within chicken hosts. Specifically, this would involve arresting the activity of virus-infected cells within the host to prevent viral replication. Rather than attempting to establish robust barriers to "prevent viral infection" as has been previously pursued, this approach renders cells vulnerable to the virus in order to prevent viral proliferation.

A promising precedent for preventing viral replication by rapidly inducing apoptosis in virus-infected cells is DRACO, reported by Todd H. Rider et al. in 2011 [8]. This research demonstrated the potential effectiveness of this antiviral concept against 15 types of RNA viruses, including influenza virus H1N1.

It is known that when RNA viruses infect cells, double-strand RNA is specifically produced during the transcription and replication processes of genomic RNA (Fig1). Influenza A virus is an ssRNA virus, but the terminal ends of its genomic RNA have complementary sequences, and this region forms a dsRNA structure. This is called a panhandle structure. DRACO utilizes the dsRNA generated during the replication process of many viruses as a marker for viral recognition and functions accordingly.

Fig 1. The life cycle of the influenza virus and its genomic RNA

During infection, influenza viruses utilize endocytosis to invade cells. Subsequently, they release genomic RNA into the cytoplasm. A partial region of this genomic RNA adopts a dsRNA structure. Typically, this virus-derived dsRNA is longer than endogenous dsRNA such as miRNA precursors [9] and has triphosphate groups at the 5' end. This region is recognized by innate immune factors such as RIG-I (Retinoic acid-inducible gene-I) [10].

Table 1. Comparison of DRACO and COCCO

Feature	DRACO	COCCO
Type	Protein Ointment	Genetically Modified Organism
Cell Membrane Permeation	Required	Not required
Recombinant Protein Purification	Required	Not Required
Human Application	Possible	Not Possible

The DRACO mechanism utilizes a fusion protein comprising a dsRNA sensor and an Apoptosis Inducer. The former incorporates the dsRNA binding domain (dsRBM) of Protein Kinase R (PKR), and the latter employs the CARD of the apoptotic induction protein APAF1.

Upon the emergence of the target dsRNA, the dsRNA binding domains of PKR undergo oligomerization, which consequently induces the fusion-linked CARD domains to also form oligomers. This molecular process subsequently triggers the activation of Caspases, thereby initiating apoptosis.

DRACO was initially developed for administration as a recombinant protein into the tissues of virus-infected individuals, and its practical application is still under development. For this reason, its components were selected for superior efficiency in recombinant protein expression and purification and for effective cell membrane penetration.

In contrast to this approach, our current project aims to create genetically modified cells that prophylactically express the protein intracellularly. This method allows for greater flexibility in the proteins we can handle and provides the ability to control their intracellular concentration. We have therefore decided to attempt system improvements and optimize the system for chickens, including testing a dsRNA sensor expected to recognize dsRNA more precisely, specifically RIG-I, and a highly effective Apoptosis Inducer, ΔCaspase9.

Fig 2. Schematic Diagram of DRACO

The dsRNA Binding Domain (dsRBD) of PKR is represented schematically by two overlapping green circles, while the Caspase Recruitment Domain (CARD) of APAF1 is shown as a yellow square. A membrane-penetrating tag such as TAT is fused to this fusion protein, enabling the protein's delivery from outside the cell.

The mechanism is as follows: The dsRBD of PKR is designed to bind to dsRNA, which promotes its dimerization (formation of a two-unit complex). Concurrently, the CARD of APAF1 is an apoptosis inducer that triggers cell death when it oligomerizes (forms a multi-unit complex).

By combining these two components into a single fusion protein, the resulting construct possesses the dual function of oligomerizing in a dsRNA-dependent manner and inducing apoptosis upon oligomerization. Consequently, DRACO can selectively induce apoptosis dependent on the presence of dsRNA.