Gene therapy approach for prevention of EAE in DA rats
T.Zargarova, O.Kulakova*, V.Prasolov*, T.Zharmukhamedova, V.Turobov, O.Derugina, M. Sudomoina*, Y.Chernaevsky**, O.Favorova*.
Branch of Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Russia; *Russian State Medical University, Moscow, Russia, and **Bone and Joint Research Unit, Barts and The London, Queen Mary’s School of Medicine and Dentistry, University of London, UK
Experimental autoimmune encephalomyelitis (EAE) as an animal model for human inflammatory autoimmune disorder multiple sclerosis (MS) is very useful for investigation of new approaches to treatment of the disease. To determine whether primary fibroblasts producing latent transforming growth factor b1 (preTGFb1) are capable of down-regulating experimental autoimmune encephalomyelitis, a retroviral vector preTGFb1 pBabe neo(-5’UTR) was used for efficient gene transfer into primary skin fibroblasts of DA rats. Intraperitoneal administration of preTGFb1-transduced fibroblasts into DA rats during the priming phase of EAE resulted in significant reduction in mortality and the mean clinical scores as compared to the control immunized animals treated with non-transduced fibroblasts.
Introduction
Autoimmune disorders such as multiple sclerosis, rheumatoid arthritis, juvenile diabetes, cardiomyopathy and other affect about 5% of the population in Europe and North America. Multiple sclerosis is an autoimmune neurological disorder mediated by antigen-specific T cells and proinflammatory cytokines, which cause demyelination of the central nervous system. Animal models are used to learn more about MS, its mechanism and treatment. EAE can be used to screen and test new drugs/methods for the treatment of this disorder. Endogenous TGFb1 is one of the cytokines protecting against Th1 cell-mediated autoimmunity, as shown by the adverse effect of neutralizing anti-TGFb1 antibodies on the course of EAE. In the present study, we have used gene therapy approach for prevention of EAE by grafting TGFb1-trunsduced syngeneic fibroblasts to rats with induced disease. The use of primary fibroblasts obtained from the host or from syngeneic subjects minimizes immunological responses of the host to the graft. These cells may be easily obtained from a skin biopsy and transduced ex vivo by retrovirus vector that integrates into the host genome, thus providing more stable therapeutic gene delivery. The aim of the present study was to investigate whether preTGFb1 gene systemic delivery using primary fibroblasts expressing latent cytokine can influence EAE course and severity.
Methods
Animals
DA/ZFV Crl BR rats (DA rats) were originally provided by Charles River Co. (Sulzfeld, Germany) and subsequently bred at the Branch of Institute of Bioorganic Chemistry (BIBCh), Pushchino, Russia. All procedures involving animals were approved by Animal Care and Use Committee of BIBCh.
Retroviral vector construction
The cDNA for human preTGFb1 was cloned into polylinker region of pBabe-neo vector. The expression of the preTGFb1 gene is from 5’- LTR promoter-enhancer region. TGFb1-pBabe-neo vector DNA was introduced by calcium-phosphate transfection into amphotropic producer line PA317, and producer cells were selected for the expression of neo by growing the cells in medium containing the neomycin analog G 418 (400μg /ml). Supernatants from G 418-resistant cells were used to infect target cells.
In vitro gene transfer
Primary fibroblasts were obtained from skin biopsy of newborn DA rat pups. Skin fibroblast cultures were maintained under standard conditions and fed DMEM supplemented with 10% heat-inactivated fetal bovine serum (Gibco, UK) three times a week. When cells reached confluence, they were passaged by trypsinization, resuspended and replated. One day before infection, producer line PA317-TGFb1 was transferred from DMEM supplemented with G418 to DMEM only. The following day, conditioned medium from producer cells was collected, filtered, mixed with polybrene (8μg/ml), and then used to infect primary fibroblasts. On the next day, the infected fibroblasts were transferred to DMEM/G418 (400 μg/ml) for selection of infected cells.
RT-PCR analysis for preTGFbeta1
Reverse transcription-PCR was used to confirm the expression of the transfered TGFb1 gene. Total RNA was isolated from cells by the GuTC method, then was transcribed into cDNA and amplified. The RNA samples were reverse-transcribed into cDNA in a reaction mixture containing RT-buffer, four dNTP (each at 1mM), 20 U of Rnasin, 5 μM random primer and 100 U of MoMLV reverse transcriptase (Promega) for 2h at 370C in a final volume of 10 μl. The resulting cDNA was amplified with 0.1U of Taq polymerase in a final volume of 10 μl, containing PCR-buffer, four dNTP (each at 0.2 mM), 0.5 pmol each of primers. PCR of cDNA was performed in parallel with primers for TGFb1 and b-actin (internal control). Primers used for the detection of preTGFb1 cDNA were: sense primer, 5’-TTGAGCCGTGGAGGGGAAATTG-3’ and antisense primer, 5’-GCCAGGACCTTGCTGTACTG-3’; for b-actin cDNA sense primer was 5'-TACTCCTGCTTGCTGATCCACATCTGC-3' and antisense primer was 5'-CATGCCATCCTGCGTCTGGACC-3'. The lengths of amplified fragments were 401 and 566 bp (for TGFb1 and b-actin, correspondingly). PCR products were separated by electrophoresis in a 2% agarose gel and visualized by ethidium bromide staining.
TGFb1 bioassay
Active TGFb1 level was determined by Mv1Lu cell line growth inhibition bioassay. Primary rat fibroblasts infected with virus from PA317-TGFb1 packaging cells were grown to confluency in DMEM, washed twice with serum-free medium and cultured for 24 h with DMEM supplemented with 1% BSA. The conditioned media were then harvested, filtered through membrane filters, heat-activated (800C, 10 min), diluted serially and added to 96-well microtiter plate (Corning Costar, USA), containing Mv1Lu cells (2x104 cells/well). Mv1Lu cells proliferation was measured using the colorimetric MTT test. Briefly, the cells were incubated with 10 μl/well MTT (5 mg/ml) for 2 h at 37°C, 5% CO2 followed by removal of the solution and the addition of 50 μl of dimethylsulfoxide. After incubation at room temperature the extinction at 550 nm was measured. The amounts of TGFb1 per ml of conditioned media were estimated using the linear part of a standard curve plotted with commercial TGFb1 (Genzyme, Cambridge, USA).
Induction of EAE and transfer of transduced cells
DA rats, weighing 220-250 g, were injected with syngeneic spinal cord homogenate in complete Freund’s adjuvant (1:1 w/v) (Difco, USA) into hind footpads (100 μl/footpad). On day 3 syngeneic primary fibroblasts transduced with TGFbeta1 gene were passaged by trypsinization, washed with medium without serum and 3x106 cells were injected intraperitoneally to each rat from experimental group. Control group consisted of immunized rats injected with similar quantity of non-transduced primary fibroblasts. All immunized rats were weighed every day and examined for clinical signs of EAE. The clinical grading was as follows: 0, asymptomatic; 1, loss of tail tonicity; 2, impaired righting reflex; 3, partial paralysis; 4, complete paralysis; 5, moribund or dead animals. The diseased animals were provided with facilitated access to food and water.
Statistical analysis
Numbers of EAE and moribund/dead animals in experimental and control groups were compared by Fisher’s exact test. The reliability of differences in mean values was estimated using the Mann-Whitney nonparametric ranking test. The p values were considered to be significant at a level of <0.05.
EAE amelioration by TGF-b1 transduced primary fibroblasts.
For the elaboration of gene therapy approach to EAE treatment we used the retroviral vector preTGFb1- pBabe-neo. The ability of primary rat fibroblasts infected with the TGFb1-pBabe-neo retroviral construct to produce and release preTGFb1 was studied in vitro. preTGFb1 mRNA expressed from TGFb1 transgene was detected in infected cells by RT-PCR (data not shown).
The biological activity of TGFb1 produced by preTGFb1-transduced fibroblasts was assessed by its antiproliferative effect on the cell line Mv1Lu (Fig.1). It is evident that TGFb1 produced by genetically modified fibroblasts, after heat activation, inhibits proliferation of Mv1Lu cells in a dose-dependent manner. Active TGFb1 contents in serum-free culture media from preTGFb1-transduced and control cells were estimated using natural TGFb1 as a standard. preTGFb1-transduced cells produced 2.4 ng/ml active TGFb1 per 106 cells per 24 h, which was 3.3-fold higher than that of the control cell.
Fig. 1 Detection of TGFb1 activity in conditioned medium from preTGFb1-transduced primary rat fibroblasts by Mv1Lu cells growth inhibition bioassay. Curves 1 and 2 – non-activated medium from control and preTGFb1-transduced cells, respectively; curves 3 and 4 – heat-activated medium from control and preTGFb1-transduced cells, respectively. log2N- logarithm of twofold serial dilutions of the conditioned media. Values are the mean ± SEM (each measurement in triplicate)
Fig.2 represents data on clinical course for three experiments pooled together and demonstrates that TGFb1-expressing cells caused a considerable decrease in the mean clinical score of disease in recipient rats compared with the animals that had been treated with the non-transduced primary fibroblasts. Most rats treated with TGFb1-transduced cells, in contrast to control animals, did not progress above grade 2 and then returned to mild clinical disability of a slight loss of tail tone, grade 0.5.
Fig.2. EAE amelioration by prophilactic grafting of TGFb1-transduced primary rat fibroblasts.
Tree days after immunization with syngeneic spinal cord homogenate in complete Freund’s adjuvant DA rats of experimental group(n=12) were treated i.p. with 3x106 preTGFb1-trunduced primary fibroblasts ( curve 1) and rats of the control group (n=10) with the same amount of non-trunduced primary fibroblasts ( curve 2). The reliability of differences in mean values was estimated using the Mann-Whitney nonparametric ranking test. The p values were considered to be significant at a level of <0.05.
This study of animal model of MS demonstrates that primary fibroblasts from DA rats genetically modified to express latent TGFb1 synthesize and release this cytokine in a latent form. Amounts of TGFb1 produced by genetically modified cells and activated in vivo at sites of inflammation are sufficient to inhibit CNS-inflammatory disease, EAE.