Design And Construction Of An Intelligent Anti-DNA Virus Immune Cell And Its Application Research

Pandemics frequently outbreak over the past few decades,and have caused serious casualties and social burdens,such as African swine fever virus(ASFV),H1N1 Flu,SARS-Co V-2,recently.Because novel virus becomes capable of spreading rapidly beyond national borders,possibly worldwide,and infecting easily from person to person.The pathogens that cause viral infectious diseases can be divided into DNA virus and RNA virus according to their genetic information.Meanwhile,the current virus detection and treatment methods in clinic are unable to achieve rapid detection and immediate elimination of virus due to limitations of external factors,such as the long evaluation period of the accuracy and the safety.Therefore,how to realize the early detection and elimination for emerging pandemic virus is still a huge challenge at present and an important scientific problem to be solved urgently.Based on engineering principles,synthetic biology designs and constructs functional elements,modules,and systems,which conduct directional design,transformation,and reprogramming of cells.Designer cells can process information and perform functions according to default demands.Designed and constructed gene circuits are also delivered into the cell,which can precisely control cell behaviors,including the controllable transgene expression,gene editing,and the precise drug release,realizing precise and controllable gene-and cell-based therapy.In this study,based on the principles of synthetic biology,we focused on infectious diseases caused by DNA virus,herpes simplex virus type 1(HSV-1)selected as a model virus for subsequent researches,and developed a set of intelligent anti-DNA virus immune cells that were integrated with detection and treatment programs to achieve point of care monitoring and controlling the behavior and movements of the virus,which provides new treatment strategies for conquering infectious diseases in the future.First,to realize the viral self-sensing,we construct an intelligent virus sensor(marked as Alice 1.0),based on the STING-mediated innate immune signal pathway,responsive to DNA virus.The fluorescence intensity of HSV-1-responsive Alice 1.0 can reach to 412.3 folds,which can be used as a virus detection device to achieve largescale,visual,and automated virus diagnosis.CRISPR / Cas9 gene editing technology can be used to knock out desired genes to eliminate virus,while the side effects of constitutive expression of Cas9,such as the risk of off-target and increased cell burden,restrict its clinical application.And highspecificity single-chain antibodies generally have unstable factors such as small molecular weight,simple structure,and short half-life in vivo,which limit their effectiveness in clinical treatment.Therefore,based on Alice 1.0,we constructed an intelligent anti-DNA virus immune cells 1.1(Alice 1.1),achieving the HSV-1-responsive Cas9 expression;an intelligent anti-DNA virus immune cells 1.2(Alice 1.2),achieving the HSV-1-responsive E317 Ab expression;an intelligent anti-DNA virus immune cells 1.3(Alice 1.3),achieving the HSV-1-responsive dual outputs,Cas9 and E317 Ab.Alice 1.1/1.2/1.3 can automatically sense the virus,and immediately exert antiviral effects.The experimental data in cellular level showed that: Alice 1.1/1.2/1.3 all displayed sensitivity to HSV-1,and had good viral elimination effects.Moreover,Alice 1.3 has the best antiviral effect in mimicking HSV-1 local infection and systemic infection in vitro,and the best antiviral effect for up to one week.The antiviral effect of Alice 1.3was significantly better than that of the clinical anti-HSV-1 drug acyclovir(ACV)lowdose medication,while achieving consistent antiviral effect with high-dose ACV medication.Alice 1.3 can avoid the risk of the emergence of drug-resistant virus strains caused by clinical single target drug and the side effects,such as renal failure,caused by high-dosage medication.In brief,Alice 1.3 can perform antiviral functions by selfsensing and elimination of virus,realizing the automated integration of virus diagnosis and treatment.To evaluate the antiviral effects of Alice 1.3 in mice,we prepared HSV-1 local infection mouse model,and transplanted Alice 1.3,capsulated with Hystem hydrogel,into mice.Evaluated from multiple aspects,such as the viral loads in the liver,spleen,and kidney of mice from the m RNA level,the expression level of Cas9 protein in the transplantation,and the expression level of E317 Ab and inflammatory factor IL-6 in the mouse serum,Alice 1.3 processes the best antiviral capability by self-sensing and elimination of HSV-1 in mice.To further excavate the antiviral effect of intelligent anti-DNA virus immune cells in mice,we prepared another mouse model for virus systemic infection.We proved that Alice 1.3 has the best antiviral efficacy by a multi-angle assessment,including the m RNA level,the protein level and the residual live virus particles level in mouse organs.In addition,we preliminarily explored the antiviral functions of Alice 1.1/1.2/1.3 in virus systemic infection mouse model.With the extension of virus infection,HSV-1-induced secreted E317 Ab can gradually act as the major antiviral force and maintain dynamic expression to inhibit extracellular free virus.Moreover,we confirm that virus can shuttle freely in the Hystem-gel-transplant to initiate Alice 1.1/1.2/1.3,assessed from the m RNA level and the protein level.Collectively,we have developed a set of intelligent anti-DNA virus immune cells equipped with self-sensing virus invasion and synchronous activation of different antiviral functional protein expression,providing new strategies for virus prevention and control in proof of concept,which may bring new hope for conquering the everchanging virus pandemics.