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Description: The objective of this research was to evaluate the efficacy of a recombinant non-viral vector for targeted delivery of a thymidine kinase (TK) suicide gene to xenograft SKOV-3 tumors. The vector was

The objective of this research was to evaluate the efficacy of a recombinant non-viral vector for targeted delivery of a thymidine kinase (TK) suicide gene to xenograft SKOV-3 tumors. The vector was genetically engineered and used to condense TK gene into particles of less than 100nm in size. The nanoparticles were used to transfect and kill SKOV-3 cancer cells in combination with ganciclovir in vitro. The results demonstrated that the vector could effectively kill up to 80% of the SKOV-3 cancer cells. In the next step, the ability of the vector to deliver TK suicide gene to xenograft tumors of SKOV-3 was studied. The results demonstrated that the vector could transfect tumors and result in significant tumor size reduction during the period that ganciclovir was administered. Administration of ganciclovir for at least three weeks post transfection was of paramount importance. These results illustrate the therapeutic efficacy and application of a designed recombinant non-viral vector in cancer gene therapy.

In the last decade, significant progress has been made in the development of vectors (viral and non-viral) for cancer gene therapy. However, none of these vectors have been able to collectively demonstrated safety, efficiency, tissue specificity, and cost effectiveness. Consequently, the need for research into innovative and novel delivery vehicles remains. Recently, there has been a great deal of interest in the development of vectors based upon biological motifs with potential applications in gene therapy. Unfortunately, none of the peptide motifs of biological origin (e.g. TAT, Mu, SV40-NLS, etc.) have demonstrated the ability to independently overcome the major cellular barriers associated with successful targeted gene delivery [1 –3 ]. In contrast, recombinant multifunctional vectors, which are the fusion of multiple biological motifs, have shown promise in overcoming these obstacles by independently performing several self-guided functions [4 –6 ]. These include, but are not limited to, cell targeting, DNA condensation, endosome disruption, and nuclear localization. Based on this information, a series of genetically engineered biomacromolecules were developed with improved efficiency, biodegradability, ease of production, and cell targetability [4. 7. 8 ]. This class of vectors (i.e. recombinant fusion vectors) not only can be used as a tool for precise structure/activity relationship studies, but holds the potential to merge the strengths of both viral and non-viral vectors in order to overcome the abovementioned obstacles[9 ].

We have recently reported the structure of a recombinant multifunctional vector with chimeric architecture composed of a HER2 targeting affibody, four repeating units of Histone H2A, and a pH-responsive fusogenic peptide (GALA) ( Figure 1 )[8 ]. We have previously shown that this vector has the ability to condense plasmid DNA encoding green fluorescent protein (pEGFP) into nanosize particles and protect from serum endonucleases, target HER2 positive cancer cells (i.e. SKOV-3) but not HER2 negative ones (e.g. MDA-MB-231), escape from the endosomal compartment into cytosol with the help of the fusogenic peptide, utilize microtubules to transfer genetic material toward the cell nucleus and mediate efficient gene expression.

Various amounts of vector GHT was dissolved in 5% glucose solution and complexed with either 1 μg of pEGFP (plasmid DNA encoding green fluorescent protein) or pSR39 (plasmid DNA encoding mutant thymidine kinase under CMV promoter). The plasmid DNA encoding SR39 gene was generously provided by Dr. Margaret Black in the Department of Pharmaceutical Sciences at Washington State University. Complexes were formed at different N/P ratios (nitrogen groups in vector to phosphate groups in plasmid DNA) and incubated at room temperature for 20 min before measurement. For example, to prepare N:P ratio of 1, 1.4 μg of vector was used to complex with 1μg of pEGFP or pSR39. In calculations of N:P ratio, we considered all positively charged N-atoms in the vector structure versus negatively charged P-atoms in pDNA. The mean hydrodynamic particle size for vector/pDNA complexes was determined by dynamic light scattering and the surface charge of particles was measured by Laser Doppler Velocimetry. All the measurements were performed using Malvern Nano ZS90 instrument and analyzed by DTS software (Malvern Instruments, UK). The particle size and zeta potential of the complexes were measured and reported as mean ± SEM, (n=3). Each mean is the average of 15 measurements and n represents the number of separate batches prepared for the measurements.

SKOV-3 cells were transfected with GHT/pEGFP complexes using the previously reported method [8 ]. In brief, SK-OV-3 cells were seeded in 96-well plates at 2 × 10 4 cells per well and incubated over night at 37°C. Cells were transfected with GHT/pEGFP complexes at various N:P ratios (equivalent of 1 μg pDNA) in McCoy’s 5A media supplemented with insulin, transferrin, selenium, ovalbumin, dexamethasone and fibronectine. The in vitro transfection studies are performed in the absence of serum. This is due to the fact that addition of serum to the media increases the viscosity of the media which could block the sedimentation of small size particles with low density (e.g. viruses or nanoparticles with sizes <200nm). Transfection in the presence of serum may be valid only for large size particle aggregates (>500nm) which have a better chance to sediment uniformly on the surface of the cells and result in transfection. For example, manufacturers of lipofectamine (Invitrogen, Carlsbad, CA) encourage users to transfect cells in the presence of Optimem where lipid particles form large size flocculates. This phenomenon is shown in reference [4 ]. To overcome this obstacle, we follow the transfection protocols for viral vectors which are targeted nanoparticles with sizes less than 100nm. For more information, please see the standard protocol of cell transfection with adenovirus from MP Biomedicals (Solon, OH).

The green fluorescent protein (GFP) was visualized after 48hrs using an epifluorescent microscope (Zeiss Axio Observer Z1) to evaluate gene expression. Lipofectamine and polyethyleneimine (PEI) 25 kDa (Sigma) were used as positive controls to validate the transfection protocol. To quantify gene expression, total green fluorescence intensity was measured using a flowcytometer (FacsCalibur, Becton Dickinson, San Jose, CA) and the data processed by FlowJo Cytometry Analysis Software. The total fluorescence intensity of GFP positive cells was normalized against the total fluorescence intensity of untransfected cells (background control). The data are presented as mean ± s.d, n=3.

In brief, 5,000 cells in control group (untransfected) were analyzed by flowcytometer to determine the fluorescence intensity of each untransfected cell. Using the 99% gating, the level of fluorescence intensity of the cell in the 99% percentile was identified and used as the basal level of autofluorescence. The fluorescence related to the top 1 percentile was considered debris. In the next step, 5,000 cells in the treatment group (transfected) were analyzed by flowcytometer to determine the fluorescence intensity of each cell. Cells that fluoresced above the basal level (established as autofluorescence) were considered transfected. The cumulative fluorescence intensity of the transfected cells is reported on the Y-axis of graphs as “Total Fluorescence Intensity”. The percentage of transfected cells was calculated by using the following formula:

% Transfection = Number of transfected cells Total number of counted cells ( i. e. 5. 000 ) × 100

To determine the expression of thymidine kinase (SR39) in cells that were transfected in vitro. a western blot analysis was performed using polyclonal antibody against thymidine kinase. SKOV-3 cells were seeded in 96-well plates at a seeding density of 2 × 10 4 cells/well. Cells were transfected with GHT/pSR39 at various N/P ratios (equivalent of 1 μg pSR39). 48 hr post transfection, cells were washed with ice-cold PBS and lysed using lysis buffer. The lysate was spun down at 12,000 g for 20 minutes at 4 °C. The supernatant was removed and protein content was determined by Bradford Assay. 30 μg of sample was loaded onto Tris-Glycine SDS-PAGE and transferred to PVDF membrane. The membrane was then blotted with rabbit anti-TK primary polyclonal antibody (R&R Rabbitry, Stanwood, WA) (1:5000 dilution) and goat anti-rabbit secondary antibody (Abcam, Cambridge MA) (1:10,000). Expression of thymidine kinase protein was then evaluated using Odyssey Infrared Imaging System (LI-COR Technology, NE). Untransfected cells were used as negative control.

To detect the expression of TK in tumors, mice that were used in protocol 9 were euthanized, tumors removed and snap-frozen in liquid nitrogen. Before use, tumor tissues were thawed on ice and dissected into smaller pieces. For each 50mg of tissue, 1.5ml ice-cold lysis buffer (50mM Tris, 150mM NaCl, 1.0% Triton X-100 with protease inhibitor cocktail) was added and homogenized with an electrical homogenizer for 15 seconds. The samples were incubated on ice for 1 hour followed by centrifugation at 12,000 xg for 20 minutes at 4 °C. The supernatant was collected and protein content was quantified using Bradford Assay. Equivalent to 30 μg of protein was loaded onto a 12.5% Tris-glycine SDS-PAGE and transferred onto a PVDF membrane. The membrane was then blotted as mentioned above and expression of thymidine kinase protein visualized. Tumors from mice that were treated with PBS used as negative control. The lysate of SKOV-3 cells that were transfected with pSR39 in vitro was used as positive control.

To evaluate the cell killing efficiency, GHT/pSR39 (equivalent of 1 μg pDNA) was administered to 1×10 4 SKOV-3 cells seeded in 96-well plates. Four hours post transfection, the media was replaced with fresh McCoy’s 5A full medium supplemented with 10% fetal bovine serum and 50 μM ganciclovir (Sigma-Aldrich, MO). Cells were incubated for a week and media supplemented with 50 μM ganciclovir was refreshed every two days. A cell toxicity assay was performed with WST-1 reagent (Roche Applied Science, IN) to determine the survival rate after the treatment. The data is reported as mean±s.d. n=3.






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