Retargeting Lentiviruses via SpyCatcher-SpyTag Chemistry for Gene Delivery into Specific Cell Types

Abstract

We report a simple strategy for the creation of lentiviral vectors specific to any desired target cells. SpyTag is inserted into an engineered Sindbis virus envelope protein and displayed on the lentivirus surface to create Sindbis virus-SpyTag pseudoparticles (Sind-SpyTag-pp). The SpyTag serves as the covalent anchoring site for a target-cell-specific cell-binding protein (CBP) that is fused to a truncated SpyCatcher (SpyCatcherΔ). Target-cell-specific lentiviruses are created by mixing the Sind-SpyTag-pp and CBP-SpyCatcherΔ in vitro We first used a HER2-binding designed ankyrin repeat protein (DARPin.9.26) as the model CBP. The DARPin-conjugated lentivirus transduced HER2⁺ SKOV3 cells with an infectious titer of 5.2 × 10⁶ IU/ml, >500-fold higher than the unfunctionalized "naked" virions (<10⁴ IU/ml). The ability of the DARPin-conjugated lentivirus to transduce HER2⁺ cells correlated with the surface expression level of HER2. Furthermore, these lentiviruses preferentially transduced HER2⁺ cells in cocultures containing HER2⁺ and HER2^- cells. To enable the use of commercially available monoclonal antibodies (MAbs) as the CBP, we developed a convenient click chemistry-based approach to conjugate MAb-derived Fab fragments to a variant SpyCatcherΔ protein containing a nonnatural amino acid, 4-azido-l-phenylalanine (AzF). Using the HER2-binding trastuzumab as a model cell-specific MAb, we created Fab-conjugated lentiviral vectors that transduced HER2⁺ SKOV3 cells with an infectious titer of 2.8 × 10⁶ IU/ml, on par with the result achieved using the DARPin-SpyCatcherΔ fusion protein. The ability to create cell-specific lentiviral vectors through chemical conjugation of a CBP should make this approach generalizable to any antibody, giving it broad utility for a wide range of research and clinical applications.IMPORTANCE Lentiviral vectors hold great potential in gene therapy. However, it remains a major hurdle to robustly engineer cell-specific lentiviral vectors. This article reports a simple and effective strategy to functionalize lentiviral vectors with cell-binding proteins, thus retargeting these viruses to cells expressing the binding partner of the CBP. The CBP is genetically or chemically linked to the SpyCatcher. The SpyTag is displayed on the virion surface as a fusion to an engineered Sindbis virus envelope protein and is used as the anchorage site for SpyCatcher-linked CBP. Using this strategy, we created lentiviral vectors highly infectious toward HER2⁺ cancer cells. The ability to rapidly create cell-specific lentiviral vectors targeting a wide range of cell types should accelerate the development of custom lentiviral vectors for many research and clinical applications.

Keywords: antibody; breast cancer; gene therapy; redirect; specificity.

FIG 1

Approach for reengineering the specificity of lentiviral vectors. SpyTag (PDB ID 4MLI [red]) is inserted into a binding-deficient fusion-competent (blinded) Sindbis virus E2 envelope protein (orange) between residues 71 and 74 and used to pseudotype lentiviruses. The CBP (e.g., DARPin; PDB ID 4J7W [dark blue]) is genetically or chemically conjugated to SpyCatcherΔ (yellow). Mixing of the SpyTag-displaying lentiviral vector and CBP-SpyCatcherΔ triggers covalent functionalization of the lentivirus with the CBP through the formation of an isopeptide bond between SpyCatcherΔ and SpyTag.

FIG 2

In vitro activity of DARPin-SpyCatcherΔ and SpyTag. One-step immobilized-metal affinity chromatography (IMAC)-purified SUMO-SpyTag (21) (20 µM) and DARPin-SpyCatcherΔ or -SpyCatcher (20 µM) were mixed in Dulbecco’s phosphate-buffered saline (DPBS) and incubated at room temperature. Samples were taken at different time points and analyzed on a 12% SDS-PAGE gel after Coomassie staining. The arrow indicates the product formed by the SpyTag-SpyCatcherΔ reaction.

FIG 3

(A) Schematic representation of chimeric Sindbis envelope protein used in this study. Both Sind-C* and Sind-SpyTag contain a 3× Flag tag at the N terminus of the protein. Sind-C* and SpyTag were inserted between amino acids 71 and 74 of the Sindbis virus E2 protein. (B) Surface expression of Sind-C* (positive control [15]) and Sind-SpyTag on 293T cells detected using mouse anti-Flag and Dylight 488-conjugated goat anti-mouse antibody. Blue, Sind-C*; green, Sind-SpyTag; red, naive 293T cells stained with the same antibodies; purple, unstained naive 293T cells. (C) Incorporation of the chimeric envelope proteins into lentiviral particles. Proteins Sind(E2)-C* (~57 kDa) and Sind(E2)-SpyTag (~54 kDa) were detected using the same antibodies used in panel B or anti-p24 antibody (41).

FIG 4

Transduction of HER2⁺ SKOV3 cells by DARPin-functionalized Sind-SpyTag-pp. (A) Bar diagram of the percentage of cells transduced with Sind-PDZ1-pp functionalized with different concentrations of DARPin-SpyCatcherΔ or SpyCatcherΔ. (B) Infectious titer of Sind-SpyTag-pp functionalized with 10 µM DARPin-SpyCatcherΔ in SKOV3 cells. Values and error bars represent the average and standard deviation, respectively, from two independent experiments.

FIG 5

Transduction of DARPin-functionalized Sind-SpyTag-pp in cells with different surface expression levels of HER2. (The estimated number of HER2 receptors per cell is shown in parentheses [32].) The naked virions and virions functionalized with SpyCatcherΔ and VSV-Gpp were included as the controls. Values and error bars represent the average and standard deviation, respectively, from 2 independent experiments. *, P < 0.05 based on Student’s t test.

FIG 6

Sind-SpyTag virions loaded with HER2-binding-DARPin proteins preferentially transduce HER2-expressing cells in cocultures. Functionalized Sind-SpyTag virions selectively transduce HER2⁺ cells in coculture. CHO-K1 (HER2-negative) and CHO-HER2-K6 (HER2-high) cells, either in isolation or at a 1:1 ratio, were transduced with DARPin-functionalized Sind-SpyTag-pp, Sind-SpyTag-pp, or VSV-Gpp at 37°C for 3 h. Forty-eight hours posttransduction, cells were analyzed for GFP and HER2 expression by flow cytometry. Representative flow cytometry plots of four independent experiments are shown.

FIG 7

Retarget lentiviral vectors with MAb-derived CBP. (A) Scheme for the creation of Fab-SpyCatcherΔ via copper-free click chemistry. (B) Transduction of HER2⁺ SKOV3 cells by lentivirus functionalized with trastuzumab-derived Fab-SpyCatcherΔ. The functionalized Sind-SpyTag-pp were serially diluted before being used for transduction. Values and error bars represent the average and standard deviation, respectively, from two independent experiments.

FIG 8

Lentiviruses surface modified via SpyCatcher-SpyTag are not inactivated by human complement. Sind-SpyTag-pp functionalized with DARPin-SpyCatcherΔ were incubated with an equal volume of untreated human serum, heat-inactivated human serum or Opti-MEM medium at 37°C for 1 h and then diluted 50-fold with Opti-MEM medium and used to transduce SKOV3 cells. The percentage of GFP⁺ cells was analyzed via flow cytometry 48 h posttransduction. Values and error bars represent the average and standard deviation, respectively, from at least three independent experiments. The difference in transduction efficiencies between samples incubated with the human serum and Opti-MEM medium is not statistically significant, with P = 0.15 based on Student’s t test.