Rationally designed synthetic vectors for therapeutic nucleic acid delivery against human cytomegalovirus infection

RNA therapy represents a great way to precisely regulate cellular processes by modulating the gene expression. Despite this potential, a profound gap exists in our knowledge of how to subsequently deliver these RNAs into the specific target cells and turn therapeutically active RNAs into practical medicines. An advanced series of interlocked, thermodynamically self‐regulated processes that enable the precise assembly of functional synthetic carriers of siRNA to the target cells in vivo was developed. To demonstrate the efficacy of this delivery system, we used it to treat human cytomegalovirus (HCMV) infection in a humanized mouse model. In this study, we use small interfering RNA (siRNA) and small complementary RNA (scRNA) to inhibit the expressions of two HCMV genes, IE1 and IE2. The auto‐regulated nanocarrier polywraplex with core‐shell structure was designed to condense and package these RNAs for delivering. To allow these particles recognize the HCMV‐infected cells, a ligand was coupled on the surface of nanoparticle, which would specifically target the HCMV‐encoded CX3CL1 chemokine receptor presented in the HCMV‐infected cells. The results demonstrated that the polywraplex conjugated with the target molecule CX3CL1 effectively and specifically delivered the siRNA/scRNA to HCMV infected cells and inhibited virus growth in vitro and in vivo.


| INTRODUCTION
RNA therapy has emerged as a potential modality that holds great promise to silence disease-causing genes with high specificity and to revolutionize the process of identifying new medicines. [1][2][3] However, turning these nucleic acids from therapeutic candidates to practical drugs is hindered by the lack of an efficient delivery system. 4,5 A therapeutically feasible nonviral carrier should accomplish five tasks of delivery simultaneously: (1) packaging nucleic acids into nanoparticles; (2) recognizing the target cells selectively; (3) escaping from endosomal digestion after cell uptake; (4) releasing nucleic acids at the intracellular site of action; and (5) metabolizing itself to biocompatible species. 6,7 This nanoparticle should also be simple in structure and easy to be formulated. 8 Although numerous gene carriers have reported and may meet some of these requirements, 9 having all the criteria to be met within a practically feasible system is of great challenging and has yet to be achieved.
We developed a series of carrier components to overcome the difficulties raised above, such as cytotoxicity induced by the carrier materials, 10 premature disassembly before reaching the target sites, 11 and the difficulty to optimize the surface population of celltargeting agents. 12 We synthesized a nucleic acid-packing cationic polymer using an endogenous molecule as the building block through pH responsive imidazole diamine backbones (pKa = 5.9) to facilitate endosomal escape and in-cytosol release of RNAs, as well as the carrier self-metabolism. 10 These natures may especially be favored for formulating RNA medicines including mRNA vaccines for their efficiency to release nucleic acids in the cytoplasm of the cells which engulf these gene-polymer particles without causing cytotoxicity. 8,10,13 We further assembled this polymer to a network structure of designable size through a unique Zeta potential regulated condensation by which small interfering RNA (siRNA) may be packed inside a single molecule of the polymer via electrostatic interactions to form size-defined carrier particles. 14 Finally, we developed a selfassembly process to enable a rationally designed tri-block copolymer to align on the surface of particle to form a functional shell to encapsulate the nucleic acid core and immobilize cell-targeting molecules in optimized surface density. 14 The shell-forming triblock copolymer possesses a multi-carboxyl-saccharide block to guide surface assembly, a hydrophobic central block forms a layer to prevent pre-phagocytic leakage of small RNAs, and a polyethylene glycol (PEG) end-block forms a steric stabilization outer-layer. The targeting agents may be conjugated to the tri-block copolymer through the selective reaction with the distal end of the PEG block and be mixed with the tri-block copolymer without the modification with desired ratio before the shell assembling. This assembly process enables the surface density of cell-targeting agents of the carrier system be precisely optimized without chemical reactions.
We call this precisely assembled core-shell structured system "polywraplex." 12 Human cytomegalovirus (HCMV) is a large DNA virus and belongs to the herpesvirus family. HCMV infection is also the leading cause of birth defects. 15 16 There is no vaccine for HCMV. Antiviral drugs are available to treat HCMV infection in immunocompromised people. These drugs include cidofovir (CDV), ganciclovir (GCV), fomivirsen, foscavir (FOS), and valganciclovir (VGCV). 17,18 Studies suggested that the development of viral resistance to the existing drugs is a major problem. Therefore, new interventions, including therapeutics development and testing, are necessary to combat HCMV infections.
In this work, we used the universal carrier system developed in our previous study 12,14 to encapsulate both siRNA and small complementary RNA (scRNA), which target the major immediateearly genes of HCMV. HCMV has two major immediate-early genes, IE1 and IE2. Both of them are essential for viral replication and are the first set of viral genes expressed at a very high level. 19,20 The

Human Cytomegalovirus Major Immediate-Early Distal Enhancer
Region is required for efficient viral replication and immediate-early gene expression. 19 Both IE1 and IE2 are encoded by the major IE locus and produced by alternative splicing. Alternative splicing of the HCMC major immediate-early genes affects infectious-virus replication and control of cellular cyclin-dependent kinase. 4 Therefore, we hypothesize that HCMV replication will be significantly inhibited if we use small RNAs to block IE1 and IE2 expressions. We chose two different methods to target HCMV IE1 and IE2 expression and designed specific scRNA to inhibit IE2 pre-mRNA splicing and siRNAs to degrade IE1 and IE2 mRNAs. These small RNAs were packaged into the polywraplex core to prevent pre-mRNA splicing and mRNA expressing [21][22][23] The activity of small RNAs will be first tested in vitro. They can be transfected into cultured cells by a wide range of methods, including physical, chemical, and biological techniques. However, the major challenge for the use of small RNAs as a biological medicine is how to deliver them into the specific target cells. We took advantages of HCMV infections in mouse model. Since HCMV will not infect any animals, we established a humanized mouse model for studying HCMV infection and pathogenesis in vivo. 24 In this model, we implanted human thymus and liver under the kidney capsule of a severe combined immunodeficient mouse to generate a severe combined immunodeficiency (SCID)-hu mouse. The HCMV carrying a luciferase marker will be inoculated in the implanted human tissue, and the infection will be monitored and measured using an in vivo imaging system (IVIS) instrument. [24][25][26] Interestingly, HCMV US28 encodes a G protein-coupled receptor and is presented on the surface of infected cells. 27 We have demonstrated that we can design a mutant chemokine molecule CX 3 CL 1 to target HCMV infected cells. 27 To deliver the small RNAs into the HCMV infected cells in vivo, we conjugated mutated chemokine CX 3 CL 1 molecule to the distal end of the PEG covalently linked with small RNA nanoparticles.
We demonstrated that this specific polywraplex system is able to successfully deliver of small RNAs to the HCMV infected cells in vitro and in vivo, and significantly inhibit HCMV replication.

| Polywraplex cytotoxicity evaluation by MTT assay
The cytotoxicity of the block copolymer and polywraplex formula-

| In vivo efficacy evaluation of polywraplex
We used SCID mice of arbitrary sex, 6−8 weeks old, to produce a SCID-hu mouse model by implanting human fetal thymic/liver tissues under the kidney capsule, as described previously, to study the therapeutic efficacy of polywraplex against HCMV. The tissues were acquired from Advanced Biosciences Resources and processed as described previously. 25 Four weeks after implantation, the implanted human tissues were surgically exposed and inoculated with 100  Figure 1B). 12,14 The successful synthesis of the polymer was confirmed by 1 H NMR (Figures 1 and 2). For the treatment of HCMV infection, a mutant chemokine molecule, CX 3 CL 1 , was selected and modified by conjugating with lysine to generate an extra amino group at its C terminus (the amino group at N terminus will be protected to avoid losing its bioactivity) (Figure 2).  Figure 2D. Then, the HS-PCL 20 -maltotriose-(COOH) n was allowed to react with modified maleimide(mal)-PEG-pentafluorophenol (F 5 C 6 ) .
The peaks in Figure 2E show the chemical shift of the methylene proton in δ3.7 ppm, which confirmed the conjugation of the F 5 C 6 -

| Formulation of the polywraplex nanoparticles
The formation of polywraplex was confirmed by observing the fluorescence images of the core and membrane, which were labeled with two different color dyes: carboxyfluorescein (FAM, green, conjugated with siRNA, complexed with polycation to label the core) and Nile Red (red, to label the hydrophobic block of the shell). 12,14 To prevent Brownian movement, the formed polywraplex was further suspended in a polyvinyl alcohol (PVA) solution and gelated after a freeze-thaw treatment. The overlay images were taken by two different incident lasers. The results showed that the green dots and red dots were nearly overlaid ( Figure 3A), indicating that a multifunctional membrane was assembled on the surface of the polyplex core. The size and Zeta potential of the nanoparticles were also characterized using dynamic light scanning measurements. The diameters of the chemokine modified nanoparticles were around 81 nm and the Zeta potential was approximately 2.61 mV ( Figure 3B). These results suggest that the synthesized polywraplex was of uniform size in the nanometer range. We concluded that after encapsulation, the extra charges on the surface of the polyplex were neutralized by the membrane.

| The functional determination of polywraplex nanoparticles
HCMV infected cells express major IE1 (72 kDa) and IE2 (86 kDa) proteins, which play critical role for transactivation of other viral genes. [28][29][30] We used scRNA to prevent the splicing of the pre-mRNA of IE1/2 and siRNA to target the IE1 and IE2 mRNA. The robustness of IE1/2 expression intervention at two independent steps represents a new therapeutic strategy to control HCMV infection. To optimize the best conditions for silencing the IE1/2 expression, the siRNA, scRNA, and IE1/2 expressing plasmid PSVH were cotransfected into 293T cells using lipofectamine 2000.
The silencing efficiency was determined by western blot analysis after 36 h of transfection ( Figure 4A). As shown in Figure 4A, WE used two types of 50 bp and nonspecific scRNA as a negative control.
The scRNA that targeted the intron region of major IE locus would prevent the splicing of pre-mRNA and reduce the expression of the

| The endocytosis of the polywraplex in HCMV-infected MRC-5 cells
To effectively target the HCMV infected cells, a chemokine molecule,  Figure 5B.
The specificity of CX3CL1-polywraplex was higher than polyplex in delivering the siRNA to HCMV-infected cells ( Figure 5B). Polyplex could deliver the siRNA-FITC randomly to infected and uninfected cells without any discrimination. We found that CX3CL1-polywraplex could deliver the siRNA to almost all the HCMV-infected cells ( Figure 5B) suggesting higher specificity of CX3CL1-polywrpalex. The enlarged merged images are included in Supporting Information: figures S1A-S1E. Our results demonstrated that CX 3 CL 1 -polywraplex could specifically target HCMV-infected cells, deliver small RNAs, and inhibit viral replication.

| MTT assay and density optimization of CX 3 CL 1
To determine the cytotoxicity of polywraplex, the 3- and IE2 protein were measured by western blot. The results showed that with the increase of the density of CX 3 CL 1 , the silencing effect of the siRNAs/scRNAs was increased. The optimized ratio was 15% to inhibit the expression of IE1/2 ( Figure 5D). Our results also demonstrated that polywraplex with specific siRNA/scRNA without the targeting molecule CX 3 CL 1 was able to The SCID-hu mice were produced by transplanting human thymus fetal tissues under the kidney capsule of SCID-hu mice. The infected SCID-hu mice were treated with the CX 3 CL 1 -polywraplex, nonspecific polywraplex, antiviral drug GCV, and saline ( Figure 6B). The viral load was determined every 24 h through total photon counts in the infected implanted tissues, which were determined using IVIS imaging of the infected animals as represented in Figure 6B,C.

|
The in vivo results confirmed that CX 3 CL 1 -polywraplex treatment ( Figure 6B) was able to reduce the HCMV infection comparable to GCV ( Figure 6B). The nonspecific polywraplex and untreated mice showed a high level of HCMV infection ( Figure 6). We observed that the HCMV in the implanted tissue could not grow till Day 14 in the case of GCV and CX3CL1-polywraplex treatment. In vitro as well as in vivo results confirmed that the polywraplex was able to recognize the HCMV infected cells due to the presence of CX 3 CL 1 and deliver the scRNA and siRNA specifically in the infected cells.

| CONCLUSION
The development of a novel small RNA or therapeutic delivery method is an imperative need for treating many human diseases.
However, the currently available methods that are in use to deliver therapeutic molecules suffer from promiscuity in targeting cells; these methods encounter difficulties in recognizing the subtypes of cells, resulting in many off-targets in terms of delivery. Therefore, the establishment of an efficient drug delivery tool for specific diseased cells will lead to the development of various therapeutic RNAs for treating infectious and metabolic diseases. To meet the requirements of RNA delivery, polywraplex, which consists of a polyplex core formed by a networked single cationic polymer nano cage condensed with gene materials and a multifunctional membrane with the targeting anchor CX 3 CL 1, was designed in this work.
We used the membrane forming block polymer PEG-PCLmaltotriose with the conjugation of carboxyl groups as a guiding block to produce assembly. The PCL block could isolate the polyplex core, and the PEG block could provide stabilization to prolong the circulation time. This nanoparticle has several advantages, such as low toxicity, smaller sizes with uniform distribution, and a quick response to low pH to facilitate endosomal escaping and degradation to release RNA after an uptake by endosomes. This precisely assembled particle could offer new modalities for gene delivery with high efficiency.
In the current study, we investigated the potential of this polywraplex system for delivering siRNAs/scRNAs to the infected cells. Notably, these polywraplexes had negligible cellular cytotoxicity, and therefore represent a safe delivery method. We demonstrated that a polywraplex nanoparticle that contained small interference RNAs was able to inhibit major viral IE gene expressions, which resulted in the substantial inhibition of HCMV replication. The polywraplex particles were loaded with scRNA that inhibited IE2 pre-mRNA splicing, in addition to siRNA, which silenced IE1 and IE2 mRNA expression.
This combinatorial approach, in which the nanoparticles were engineered to have both molecules, resulted in the complete inhibition of viral replication. This advancement in nanotechnology specifically targeted the infected cells that expressed a highly specific CX 3 Cl 1 molecule and were bound to the HCMV encoded US28 proteins. Therefore, our highly efficient delivery technology, conjugated with more specific target molecules, resulted in a robust tool to inhibit HCMV replication. We also tested the polywraplex system in delivering the siRNAs/scRNA in an in vivo model system using SCIDhu mice. Polywraplex was highly effective in inhibiting HCMV growth in implanted thymus tissues as compared to a control. These results suggest the great therapeutic potential and efficacy of polywraplexbased nanoparticles in treating viral diseases. Thus, polywraplex has immense potential for delivering various therapeutic molecules, including, but not limited to, siRNA, scRNA, drugs, vaccines, proteins, mRNA, and DNA, for the treatment of various diseases.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.