Title:Tet-R regulated gene delivery to enhance wound healing
Authors:Nicola C Petrie MB ChB, Jan J Vranckx MD, Daniela Hoeller MD, Patrik Velander MD, Christoph Theopold MD, Feng Yao PhD, Elof Eriksson MD
PDGF is a recognised vulnerary agent, which demonstrates chemotaxis for fibroblasts (1), macrophages, neutrophils and smooth muscle cells (2). In-vitro studies have shown that it can stimulate the proliferation of fibroblasts (3), smooth muscle cells (4), endothelial cells (5), (6) and glial cells (7) functioning by both autocrine and paracrine mechanisms. These specific in-vitro characteristics imply an in-vivo physiological role of PDGF in the stimulation of cell regeneration in response to tissue damage – a hypothesis further supported by numerous animal studies. Initial investigations were performed using recombinant proteins and although several investigators have delivered rPDGF-B to incisional wounds and elegantly demonstrated an increase in breaking strength, such findings lack relevance to excisional wounds, which represent the majority of wounds in need of specialist treatment (8). Recombinant PDGF-BB when delivered to excisional wounds has been reported to increase granulation tissue thickness (9), inflammatory cell influx, extracellular matrix deposition (10) and rates of wound closure (9). The delivery of recombinant proteins to wounds, however, suffers from a number of shortcomings including short half-life and poor bioavailability from the delivery vehicle, which necessitate frequent administration incurring high costs. Gene therapy approaches to growth factor delivery have overcome many of these problems, for example, continued release avoids erratic fluctuations in protein concentration and frequency of administration. Wound healing gene therapy using PDGF proteins has been described in a number of studies, ususally by way of adenoviral constructs. The theoretical concerns of viral recombination and host immune response remain serious threats and highlight the need for developing safer methods of gene transfer.
Here, we report the development and testing of a porcine cell line, which over-expresses PDGF-BB for sustained periods in-vivo. When tested in either autologous or allogenic recipients, the effects on wound healing were comparable, a finding which supports the use of pre-cultured allogenic cell lines as vectors for gene therapy. Establishment of optimal levels of transgene expression both through accurate quantification and assessment of effect is a prerequisite to the implementation of methods capable of delivering the desirable dose. This tailoring of expression has been enabled by the development of genetic switches, first reported using the prokaryotic lac-repressor gene (from Escherischia coli) (11), and subsequently using the E.coli-derived tetracycline-repressor (tet-R) gene to demonstrate expression in eukaryotic plant cells (12). Gossen and Bujard’s seminal paper (13) describing the combination of a tet-R fusion protein with the tet-Operator (tet-O) containing hCMV promoter was critical to the development of biological systems to control gene expression in mammalian cells. Yao et al (14) describe a modification of this design, which through accurately positioning tet-R alone thereby avoiding the use of a fusion protein, and inserting two copies of the tet-O gene, complications such as cellular toxicity and transcriptional quelching (15) can be avoided, whilst enabling the tightening of transcriptional control (16). Using Yao and colleagues’ T-Rex™ system, in this study we demonstrate the development of a porcine fibroblast cell line expressing PDGF-BB under the control of a tetracycline-regulated genetic switch. Furthermore, we provide evidence for effective, regulatable expression of PDGF-BB both in-vitro and when transplanted in-vivo in a porcine model of wound healing. This model offers a standardised method of regulating and quantifying transgene expression in a full-thickness wound model and highlights the importance of determining and controlling growth factor delivery by demonstrating the deleterious effect which supraphysiological doses of PDGF-BB have on wound healing. In conclusion, the future of gene therapy research, whether for the treatment of wounds or other applications, will rely on such controlled, reversible systems to ensure efficient, predictable, safe methods of transgene expression.
Methods:
The human PDGF-B cDNA-containing Eco RI fragment in plasmid pMT2 (a kind gift from Dr Charles Stiles, Dana-Faber Cancer Institute, Boston, MA) was subcloned into the Eco RI site of the pcDNA4TO plasmid (Invitrogen, CA). pcDNA4TOPDGF-BB is a recombinant plasmid in which PDGF-B is under the control of the tet-Operator bearing CMV promoter confirmed by DNA sequencing using a hCMV forward primer (Invitrogen, CA). For the development of a PDGF-BB expressing stable cell line skin was harvested from ear clippings from piglets taken routinely by the animal farm staff within 4 hours of birth and a dermal fibroblast cell line isolated. Fibroblasts in 6th passage were co-transfected 24 hours following seeding (at 1x106 cells/dish) with pcDNA4TOhPDGF-BB (9.5 ug) and pcDNA3 (0.5 ug) (encoding a Neomycin resistance gene) (Invitrogen, CA) using 40l lipofectin (Invitrogen, CA) in 500l OptiMEM (Gibco, NY). At 72 hours cells were re-seeded and selected in medium containing 400g/ml Geneticin (G418 – Sigma, MO). Resistant colonies were expanded and a hPDGF-BB ELISA (R&D, MN) performed on supernatant to confirm PDGF-BB expression. The cell line expressing highest levels of PDGF-BB over successive passages was chosen as the treatment cell line and a corresponding G418-resistant cell line expressing only basal levels of PDGF-BB was chosen as the control cell line for further experiments. For the development of a double stable cell line expressing PDGF-BB under the control of the Tetracycline switch previously established G418-resistant hPDGF-BB expressing cells were seeded at 300,000 cells/dish into 60mm culture dishes. Transfections were performed after 24 hours using 6g pCEP4 DNA (encoding TET-R and Hygromycin resistance) and 24 l Lipofectin (Invitrogen, CA) and confluent cultures of pCEP4 transfectants passaged after 96 hours. Cells were cultured in DMEM supplemented 50g/ml Hygromycin B and individual colonies selected. The cell line demonstrating highest regulation of induceable hPDGF-BB expression was chosen for subsequent experimentation. Further in-vitro testing was performed on this cell line to confirm the degree of regulatable expression with time and increasing dose of tetracycline. All animal protocols complied with the current criteria for humane care of animals and were approved by the Harvard Standing Committee on Animal Research. A total of four Yorkshire pigs were used, two to test the stable cell line (one as an autologous transplantation in its own donor and one to test allogenic transplantation) and two to test the effect of regulated expression from the stable cell line. The animals were starved prior to surgery and sedated with a mixture of 100mg Xylazine (Phoenix pharmaceuticals Inc, MO) and 300mg Ketamine (Abbot Laboratories, IL). Anesthesia was induced and maintained using a mixture of Isoflurane (0.8-4%) and oxygen (3.5L/min) via an endotracheal tube on the first day and via a snout mask thereafter and continuous peri-operative monitoring of oxygen saturations, pulse rate and temperature performed. The pig was depilated by waxing and the skin prepared using 10% Povidone Iodine and 70% Isopropyl alcohol. For each pig, a total of 20 full-thickness wounds measuring 2.5x2.5cm were traced, tattooed and excised to a depth of 1cm under sterile conditions. Hemostasis was achieved and the wounds enclosed by vinyl chambers. Meanwhile in-vitro cells were harvested and counted prior to re-suspension in DMEM with antibiotics (100u/ml penicillin and 100g/ml streptomycin) at a concentration of 300,000 cells per 3ml per wound. Wounds were randomized over the pigs’ dorsa to negate any healing bias conferred by position. Wounds on the two pigs used to test the stable cell line were injected with 3ml of either 300,000 PDGF-BB expressing cells or 300,000 control cells or 3ml DMEM with antibiotics (no cells). The allogenic pig was treated with an additional treatment group consisting of 270,000 control cells and 30,000 PDGF-B expressing cells to determine whether a 10-fold lower number of cells correlated with a 10-fold lower level of PDGF-BB expression and what effect this may have on healing. Pigs used to test the effect of regulated expression from the stable cell line received 3mls with 300,000 cells suspended in either 0, 100 or 2,500ng/ml tetracycline or 3mls of DMEM (no cells). Anesthesia was maintained for a further 2 hours following the injection of cells to allow them to settle after which it was reversed and 300g Buprenex (Reckitt Benciser Pharmaceuticals Inc, VA) given for post-operative analgesia. Cells for in-vitro controls were prepared at the time of operation by seeding equivalent numbers (300,000) of cells from each treatment group into 60mm culture dishes and used to confirm continued proliferation by microscopic evaluation of cell density and PDGF-BB expression in the supernatant. 1 ml of supernatant was removed from each dish every 24 hours and stored at –80° for use in the same ELISA that would eventually determine in-vivo expression (R&D, MN). The remaining medium was removed and replaced and this procedure continued until cells became confluent. Daily follow-up measurements
consisted of wound fluid harvest on a daily basis, which was collated according to treatment group and stored at –80°C for later quantification of PDGF-BB concentration. Wound chambers were removed, the skin cleaned, new chambers reapplied and 3ml Saline with antibiotics (100u/ml penicillin and 100g/ml streptomycin) with or without tetracycline as appropriate injected. Wound contraction measurements were made every third day by planimetry and analyzed using Scion Image Software. Cross sectional, full-thickness wound biopsies were harvested on the 12th post-operative day after which the pig was euthanized using 10ml Euthasol solution (Delmarva Laboratories Inc, VA). Wound biopsies were fixed in 4% formalin, paraffin embedded, cut and stained with Haematoxylin and Eosin or Masson’s trichrome. Histological specimens were analyzed for inflammatory response and scanned into digitized images for linear measurement of re-epithelialisation using Paint Shop Pro software.
Results:
Results for both pigs receiving the PDGF-BB expressing cell line provide evidence of significantly higher levels of PDGF-BB in exudate compared to those treated with either G418-resistant non-expressing cells or saline alone. In the allogenic pig expression peaked at day 7 with 7,277pg/ml compared to day 4 in the saline controls (400pg/ml) then declined but remained above baseline expression until the end of the experiment at day 12. No significant difference between PDGF-BB levels in wounds treated with saline or the control cells was observed. In-vivo expression in the autologous pig demonstrated a similar profile of secretion but with higher values possibly reflecting greater cell survival. Peak expression was seen between days 5 and 7 when levels exceeded recordable values (>20,000pg/ml). Again, levels remained elevated until the end of the experiment at day 12 (1,346pg/ml) compared with saline and control cell line groups (32pg/ml and 31pg/ml respectively). Both saline and control cell line treatments caused a peak in PDGF-BB expression at day 9 (421pg/ml and 363pg/ml respectively) and demonstrated no significant difference in levels of expression throughout the procedure. In both of these pigs results for re-epithelialisation demonstrated that wounds treated with the PDGF-BB expressing cell line demonstrated a statistically significant delay in rates of re-epithelialisation from 71% to 54% in the allogenic animal (p<0.05) and from 72% to 42% in the autologous pig (p<0.05). Results of in-vivo expression from the two pigs in which the gene switch cell line was tested confirmed regulatable expression of PDGF-BB. The saline group reveals background levels of expression, which vary very little, peaking at day 3 with 274pg/ml and dropping to baseline values by day 6. Wounds in the TET-0 group (wounds transplanted with cells but receiving no tetracycline) demonstrated levels, which were significantly higher than the saline group thereby confirming in-vitro findings that PDGF-BB expression in this cell line is not fully repressed by the tetracycline switch. Expression in this group peaked at day 3 with 3,228pg/ml PDGF-BB. A dose dependent increase in PDGF-BB expression was seen as tetracycline doses increased. The maximum expression in the TET-150 group was 4,633pg/ml, which peaked at day 4 and in the TET-2500 group was 6,023pg/ml, again at day 4. Rates of final re-epithelialisation for grouped wounds, calculated as a linear percentage of the initial wound measurement were 37%, 39%, 38% and 37% for the saline, TET-0, TET-150 and TET-2500 groups respectively. All values had equivalent standard deviations and there were no statistically significant differences.
Conclusions:
We conclude that high-level PDGF-B transgene expression in full-thickness wounds is achievable using non-viral methods utilizing both allogenic and autologous cell lines. We also conclude that over-expression of a vulnerary agent may be deleterious and highlight the need for developing methods to accurately measure and regulate transgene expression in-vivo thereby increasing efficacy and safety of current gene therapy strategies. We have demonstrated the development and use of a stable cell line incorporating a genetic switch, capable of regulated PDGF-BB expression in-vivo. We have highlighted the importance of imposing such control in gene therapy studies and of optimising quantitative measures for accurately monitoring levels of transgene expression achieved. Before gene therapy for PDGF-BB, or any other growth factor can be used clinically, more information is required. Precise delineation of therapeutic ranges must be established and systems designed to ensure delivered doses remain therein. Once these basic safety issues have been addressed, gene switches offer further potential by enabling a more physiological delivery of vulnerary agents by timing expression of transgene products to mimic the clinical situation and by combining a number of agents with overlapping profiles.
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Figure 1:Schematic representation of the Tet-R switch.
Cells must contain both the tetracycline operator (Tet-O) and tetracycline repressor (Tet-R)sequences. In the absence of tetracycline (A), the repressor binds to the operator thereby inhibiting downstream transcription. In the presence of tetracycline (B), the repressor is released thereby enabling transcription to occur unimpeded.