Therapeutic protein transduction of mammalian cells and mice by nucleic acid-free lentiviral nanoparticles

Abstract

The straightforward production and dose-controlled administration of protein therapeutics remain major challenges for the biopharmaceutical manufacturing and gene therapy communities. Transgenes linked to HIV-1-derived vpr and pol-based protease cleavage (PC) sequences were co-produced as chimeric fusion proteins in a lentivirus production setting, encapsidated and processed to fusion peptide-free native protein in pseudotyped lentivirions for intracellular delivery and therapeutic action in target cells. Devoid of viral genome sequences, protein-transducing nanoparticles (PTNs) enabled transient and dose-dependent delivery of therapeutic proteins at functional quantities into a variety of mammalian cells in the absence of host chromosome modifications. PTNs delivering Manihot esculenta linamarase into rodent or human, tumor cell lines and spheroids mediated hydrolysis of the innocuous natural prodrug linamarin to cyanide and resulted in efficient cell killing. Following linamarin injection into nude mice, linamarase-transducing nanoparticles impacted solid tumor development through the bystander effect of cyanide.

Figure 1

Molecular configuration of PTNs. (A) Schematic representation of expression vectors required for the protein transduction technology. Transgenes are drawn to scale. (B) Illustration of production and use of PTNs. (1) Transient cotransfection of (i) helper construct pWW203, (ii) pseudotyping vector pLTR-G, (iii) heterologous protein-encoding lentivector (pPOI) into the PTN production cell line HEK293-T. (2) While pWW203- and pLTR-G-encoded proteins mediated assembly of nucleic acid-free pseudotyped HIV-1-derived lentiviral nanoparticles (2a), the VPR-PC-tagged protein of interest (POI) was produced (2b) and targeted to the lentivirions (2c), where the VPR-PC tag was released by the pol-encoded viral protease and the native POI was encapsidated into budding PTNs (2d). (3) PTNs recovered from the culture supernatant were purified and concentrated by centrifugation and administered to target cells into which the POI was transduced (4). Abbreviations: 5′LTR, 5′ long terminal repeat; GAG, HIV-1 gene encoding core proteins; GFP, green fluorescent protein; HSVtk, Herpex simplex virus type 1 thymidine kinase; LIS, Manihot esculenta (Cassava) linamarase; pA, polyadenylation site; P_hCMV, human cytomegalovirus immediate early promoter; P_hEF1a, human elongation factor 1a promoter; RIPDD, RIP death domain—C-terminal domain of the human serine-threonine kinase 1 receptor; POL^(*), gene encoding virion-associated enzymes including [*] integration-deficient integrase; VSV-G, vesicular stomatitis virus G protein; VIF/VPR/REV/TAT/VPU, HIV-1 accessory proteins; VPR, encapsidation-competent HIV-1-derived accessory protein; PC, pol-derived protease cleavage site.

Figure 2

Localization and encapsidation of VPR-PC-tagged heterologous proteins in HEK293-T producing PTNs. (A) Intracellular distribution of VPR-PC-tagged and native GFP (scalebars 100 µm). (B) Fluorescence micrographs of pellets obtained by centrifugation of HEK293-T supernatants transfected with helper plasmids pWW203 and pLTR-G and (i) pNLK9 (P_hEF1a-VPR-PC-GFP-pA; 12 µg p24), (ii) pNLK8 (P_hEF1a-GFP-pA; 17 µg p24) or (iii) no additional vector, (12 µg p24) (scalebar 500 µm). (C) Western blot analysis of PTN-producing cell lysates (i) and HIS- (ii) or HA- (iii) tagged proteins in centrifugation-purified PTN preparations equally adjusted to 135 ng p24. VPR-linked as well as processed native proteins are visualized (GFP, 28 kDa; RIPDD, 16 kDa; Herpes simplex thymidine kinase [HSVtk], 37 kDa, Linamarase [Lis], 75 kDa). The 50 kDa signal in (iii) represents an unspecific interaction between a PTN protein and the HA tag-recognizing antibody.

Figure 3

Impact of PTN-delivered linamarase-linamarin prodrug system on (tumor) cell viability 96 h after administration. (A) Light microscopy of monolayer cultures treated with (i) linamarase-transducing nanoparticles (PTN_LIS) and linamarin, (ii) PTN_LIS only, (iii) linamarin only or (iv) empty control particles (scalebar 50 µm). (B) Viability of cells treated with PTN_LIS and linamarin (Lin), Lin or PTN_LIS alone or untreated as control (Cntrl).

Figure 4

Cyanide production in CHO-K1 treated with a combination of linamarase-transducing nanoparticles (PTN_LIS) and linamarin. (A) Correlation of PTN_LIS number administered and the cyanide concentration produced in CHO-K1 cultures after 72 h. (B) Cyanide production kinetics in PTN_LIS-transduced CHO-K1 cultures (5 × 10⁸ PTN's, 800 000 cells, 8 ml culture medium).

Figure 5

Impact of PTN-delivered linamarase-linamarin prodrug system on 4T1-derived tumor spheroids. (A) Light and fluorescence (ethidium bromide, dead cells shown in red; fluorescein-diacetate, viable cells shown in green) micrographs of 4T1-derived tumor spheroids treated up to 120 h with linamarase-transducing nanoparticles (PTN_LIS) plus linamarin, PTN_LIS alone, NaCN or mock-transfected PTN production supernatants as control (Mock) (scalebar 200 µm). (B and C) Quantitative analysis of 4T1- and MCF7-derived tumor spheroid diameters changes (B) and viability (C) 120 h after administration of PTN_LIS/LIN, PTN_LIS, NaCN or linamarin as control (Lin).

Figure 6

Tumor growth retardation in mice after administration of linamarin-transducing nanoparticles (PTN_LIS) and linamarin (Lin). (A) Size of 4T1 tumors and (B) serum cyanide levels of mice 4 days after injection of PTN_LIS and Lin, PTN_LIS alone, NaCN or Lin alone (Cntrl). (C) Histologic analysis of treated tumor explants using haematoxylin–eosin (i,ii,iii and iv; sections; scalebar 400 µm) and toluidine blue (i′,ii′,iii′ and iv′; semi-thin sections; scalebar 20 µm) staining revealing the effects of different treatment modalities versus control in 4T1 tumors. i and i′: Tumors treated by a combination of PTN_LIS and linamarin showed a large necrotic area around the application site. Black triangles indicate apoptotic figures and bodies at different stages of chromatin consolidation. ii and ii′: Treatment with PTN_LIS alone had practically no effect on the tumor growth. The blood vessels were preserved (black circles) and mitotic figures (black diamonds) were detectable close to the application site. iii and iii′: Sodium cyanide injection had a pronounced effect only locally at the site of application as indicated by apoptotic profiles (black triangles). At a distance lower than 250 µm of the application canal tumors exhibited normal cell morphology (black squares). iv and iv′: The tumor cells in the control group treated by Lin demonstrated typical mitotic activity (black diamonds) and normal blood vessels (black circles) were formed.