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Duchenne muscular dystrophy (DMD) is a genetic disorder that is characterized by the lack of the protein dystrophin which induces muscle wasting. The disorder is common among males and does not have a cure. Genetic engineering and stem cells provide an interesting therapeutic potential for alleviating DMD. Zinc finger nucleases are specifically engineered enzymes that are used to genetically modify muscle-derived stem cells (MDSC) to express dystrophin- these MDSCs are transplanted into SCID-mdx mice. This work contains the current isolation, gene targeting and transplantation techniques of MDSCs that provide a plausibility to ameliorate the disorder.
Duchenne Muscular Dystrophy (DMD) is a destructive X-linked (Huard et al., 2003) recessive genetic disorder typified by mutations in the dystrophin gene that disrupt the reading frame of the dystrophin mRNA (Benchaouir, et al., 2007; Peng and Huard, 2004). This gene encodes a protein that is essential for maintaining the structural integrity of the myofibres, therefore, the absence of the functional gene cause the myofibres to undergo fibrosis rather than regeneration (Peng and Huard, 2004). The mutation that causes DMD can be rectified using gene targeting. The mutation is replaced with a functional gene using Zinc finger nucleases (ZFN) that removes the mutation and modulates homologous recombination of the functional gene which is introduced (Porteus and Carroll, 2005). Transplantation of genetically altered stem cell progenitors that can differentiate into skeletal muscle and also produce the functional dystrophin protein presents a therapeutic approach to treat DMD. One such stem cell progenitor that is used is muscle-derived stem cells (MDSC).
MDSCs are adult stem cells residing in skeletal muscles (Torrente et al., 2001) and are characterized by a maker profile-Sca-1, CD34+/-, c-kit -, CD45-, desmin+, CD13+, CD56+ and Bcl-2+ (Jankowski, 2002; Danisovic et al, 2008; Péault et al., 2007).The Sca-1/34+ cells have higher proliferative capacity than the Sca-1/CD34- cells and also express the myogenic marker, desmin (Charge and Rudnicki, 2004).The skeletal muscle tissue provides a good source for isolating these pluripotent stem cells (Lee, et al, 2000) which on intraarterial injection have the capacity to migrate to the muscle and bring about myogenesis (Torrente et al., 2001). Therefore, MDSCs are an efficient target for gene modification. Viral or non-viral vectors are used to deliver the full length donor gene or one of the many mini-dystrophin genes into the stem cells which is then introduced into SCID-mdx mice and checked for dystrophin production. (Benchaouir, et al., 2007)
Aims and Objective:
To isolate human MDSCs, genetically modify the mutation causing DMD using ZFN and its transplantation into SCID-mdx mice. To investigate whether the genetically modified MDSCs can differentiate in vivo and produce dystrophin when transplanted.
Program of Research:
. Culture of MDSCs: Isolation of MDSCs by Modified Pre-plating Technique
Tumor-free skeletal muscle sections (or biopsy) are obtained from patients, on informed consent, after surgery. The muscle mass obtained is rinsed with sterile phosphate buffered saline (PBS) (Danisovic et al., 2008), minced and digested enzymatically using 0.2% collagenase- type XI for 1 hour at 37ËšC followed by 0.1% trypsin-EDTA for 30 min at 37ËšC. Filter the cells using a 70µm filter and centrifuge at 1000 rpm for 5 min. The supernatant is discarded and the sediment is resuspended into cell culture dishes containing DMEM (Dulbecco's modiï¬ed Eagle's minimal essential medium) (Lu et al ,2009; Danisovic et al, 2008; Qu-Petersen et al,1998) supplemented with 10% fetal bovine serum, 10% horse serum, 1% penicillin-streptomycin, and 0.5% chick embryo extract (Jankowski et al.,2001). The culture dish is then placed in a CO2 incubator (37 °C, 5 % of CO2) for 1 hour (preplate1, PP1). The non-adherent cells in suspension are removed and transferred to a fresh flask and incubated for 2 hours (PP2) (Qu-Petersen et al, 1998; Jankowski et al., 2001) and the medium in the first flask in replaced. Serial replating with the supernatant for PP3-6 is carried out at a 24 hour period. The late pre-plate cells (PP6) are found to be more efficient in differentiating into muscle cells and also have a higher survival capacity once transplanted (Torrente et al., 2001)
Characterization of MDSCs
Immunohistochemistry of MDSC is performed to check for the presence of the stem cell markers and hence, it differentiation potential. The cells were fixed with cold methanol for 1 min followed by immunofluroscent staining against α-actin and desmin (Danisovic et al, 2008) to indicate the expression the α-actin and desmin.
The cells are stained for the characteristic markers-Sca-1, c-kit, CD34 andCD45. The cells from the culture dish are removed using trypsin-EDTA solution (0.25% trypsin-2.6mM EDTA). Centrifuge and wash cells in cold PBS solution containing 5% BSA and 0.1% sodium azide. The cell suspension is divided into 4 aliquots: One control and 3 tubes with combinations of the monoclonal antibody. Fetal calf serum (1:10 dilution in PBS) and Fc block are added to each tube, 10 min on ice. Optimal amounts of monoclonal mouse anti-human antibodies (Sca-1, CD34, c-Kit, and CD45) (Lu et al, 2009) are added directly to each tube, 30 min. FITC-conjugated anti-CD45 antibody is added to the non-control tubes with one of the following combinations of monoclonal antibodies: (i) R-PE-anti-Sca-1 and biotin-anti-CD34, (ii) R-PE- anti-CD117 (c-Kit) and biotin-anti-Sca-1, and (iii) R-PE-anti-CD117 and biotin-anti-CD34 antibodies. The control tubes receive equivalent amounts of FITC-conjugated, biotin-conjugated, and R-PE-conjugated isotype antibodies. Wash each tube in cold PBS solution and centrifuge. Streptavidin-allophycocyanin (APC) conjugate is added to the pellet in all four tubes and incubated on ice for 20 min followed by washing. Prior to analysis, 7-amino-actinomycin D (7-AAD) is added to each tube to exclude dead cells. A minimum of 10,000 live cell events is collected and the phenotypic characterization is displayed (Jankowski et al., 2001)
. Gene therapy:
DMD is characterized by monogenetic mutations and hence, can be corrected using Zinc finger nuclease (ZFN) therapy. The ZFN are customized nucleases and can be made to target the mutation in the dysfunctional dystrophin gene. ZFNs function by specific binding to the target gene. Once bound it brings about a double stranded cut in the gene followed by its 5'exonuclease activity thus, deleting the mutation. Mini-dystrophin gene or the entire functional portion of the gene from the donor that is introduced undergoes homologous recombination with the target gene to restore a functional dystrophin gene. The method used here follows a variation from previous work (Benchaouir et al., 2007; Urnov, et al., 2005; Cathomen and Joung, 2008) in which the exon mutations present in the DMD gene of the patient are targeted. The cultured MDSCs from PP6 are used as they render a high degree of transgene expression, survival and fusion with the myofibres after transplantation (Torrente et al., 2001; Qu-Petersen et al, 1998). The ZFN is constructed with two DNA-binding domains, each containing four zinc-ï¬nger motifs that can recognize a total of 24 base pairs specific to the nucleotides around the 'mutation hotspot'. The DNA-ZFN can be optimized, if required, to enhance binding and cleavage. The ZFN specific to the patient's mutation is engineered into expression plasmids and is transfected in two concentrations (Urnov, et al., 2005) into the cultured MDSCs followed by introduction of the viral vectors (e.g.: adeno-associate vector, AAV) containing the donor derived homologous gene with the correct sequence of the dystrophin or mini-dystrophin gene (Cathomen and Joung, 2008; Porteus and Carroll, 2005).
The MDSCs that were transduced with the gene are cultured in differentiation medium, F12 (with 10% FCS, 5% horse serum, 0.1µM dexamethasone, 50µM hydrocortisone and 1% penicillin/streptomycin) for about 14-28days. The cells are then checked for the production of dystrophin mRNA using RT-PCR and DNA sequencing (Porteus and Carroll, 2005; Lu et al., 2009b; Benchaouir et al., 2007).
Analysis of the cultured cells using RT-PCR:
Total RNA is isolated and Reverse transcription (RT) is executed for the first strand of cDNA followed by PCR amplification of the gene product using Taq polymerase and primers designed for dystrophin gene. The PCR products are checked by agarose gel electrophoresis (Lee et al., 2000).
. Intraarterial transplantation of the genetically modified MDSCs into SCID-mdx mice
The genetically engineered MDSCs are introduced intra-arterially into the muscle in SCID-mdx mouse (mdx mice is a model for DMD which has point mutation in the dystrophin gene) to 'repopulate the diseased tissue'. Once the cells are injected they circulate and bind to the endothelium of the muscle capillaries and migrate to the muscle and aids in myogenesis and release the functional dystrophin (Torrente et al., 2001). The damaged muscle in DMD causes the release of various cytokines, chemokines and other intracellular proteins that are involved in inflammation that induces the expression of chemoattractive receptors. This is shown to mediate the 'homing' on the stem cells to the affected region and can regenerate the muscle tissue and also produce dystrophin (Péault et al., 2007). After 21-45 days muscle from the SCID-mdx mice is removed and the cells are characterised using immunohistochemistry. The MDSCs potential to differentiate into muscle fibres and produce dystrophin is checked by detecting the mRNA expression using PT-PCR (Benchaouir et al., 2007).
Immunohistochemistry of the muscle:
Dystrophin that is produced in the transplanted SCID-mdx mice is examined by immunofluroscent staining of the muscle sections with specific anti-dystrophin antibodies (anti- Dys3). Muscle samples are removed and frozen in liquid nitrogen-cooled isopentane and fine sections of the muscle are taken and incubated with anti-Dys3 overnight at '4Ëš C in PBS supplemented with 1% BSA and 0.2% TritonX-100' (Dellavalle, 2007).
The total RNA is isolated as described above by Lee et al., and is used for detection of functional dystrophin mRNA.