Effects of manipulating the immune system on dystrophin gene transfer and dystrophic phenotype in striated muscles of Duchenne muscular dystrophy model, mdx mouse

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by mutations in the gene coding for dystrophin protein, which give rise to a dysfunctional protein in skeletal muscle. Dystrophic muscle progressively degenerates. In addition, necrotic muscle fibers undergo high levels of inflammation that in turn promote the pathology that is associated with this devastating disease. Therefore, treatments that 1) restore expression a functional dystrophin protein in dystrophic muscles, and 2) lower the ongoing inflammation in the necrotic muscle tissue, are both important in ameliorating DMD phenotype. Transfer of a functional dystrophin gene using a viral vector can help restore the missing dystrophin protein in dystrophic muscles. The host immune system, however, is a major barrier to successful vector-mediated dystrophin protein expression in a dystrophic host, as anti-dystrophin immune response leads to rejection of the protein. Here I show that temporal elimination of the host immune system by irradiation in the mdx mouse, a murine model of DMD, prior to vector-mediated dystrophin gene delivery, leads to a delayed and diminished host anti-dystrophin immune response. These findings are important for a better evaluation of anti-dystrophin immunity in a dystrophic host. In the case of lowering inflammation in dystrophic muscles, I investigated the effects of rapamycin, a potent immunosuppressant, on both dystrophic phenotype and dystrophin gene transfer in mdx mice. Treatment of adult mdx muscles with rapamycin lead to significantly lower levels of muscle fiber necrosis and reduced effector T cell infiltration in dystrophic muscles. These events correlated with a difference in activation of the mammalian target of rapamycin (mTOR) in the diaphragm muscle, but not the TA muscle, suggesting a differential regulation of mTOR activation in the two tissues. Rapamycin treatment, however, did not allow for a higher level of vector-mediated dystrophin protein expression in treated muscles. In general, these findings shed more light on the effects of manipulating the immune system in a dystrophic host in terms of both reducing the inflammation that is associated with DMD and reducing anti-dystrophin responses following gene therapy, suggesting that regulation of the immune system is essential in ameliorating DMD

Cloning and Expression of Human Membrane-Bound and Soluble Engineered T Cell Receptors for Immunotherapy

We report here the design and construction of several gene vectors for expression in mammalian cells of membrane-bound and soluble human T cell receptors (TR). We designed a vector (TR-ALPHA-IRES-TR-BETA pEF4) that encodes high-level expression of the full-length TR on the surface of T cells. Furthermore, we engineered TR that does not require the presence of endogenous CD3 molecules for surface expression and thus expression is not limited to T cells. We also constructed a vector encoding a single-chain TR (scTR) as a fusion protein of V-ALPHA-V-BETA-C-BETA with CD3Z. Since it is encoded and expressed as a single molecule, this scTR is well suited for gene therapy. Lastly, we successfully used a mammalian expression vector for generation of soluble human TR. The approaches we used here for manipulation of a human tumor-specific TR can be useful for other investigators interested in TR-based immunotherapy

Effect of rapamycin on immunity induced by vector-mediated dystrophin expression in mdx skeletal muscle

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene. Therapeutic gene replacement of a dystrophin cDNA into dystrophic muscle can provide functional dystrophin protein to the tissue. However, vector-mediated gene transfer is limited by anti-vector and anti-transgene host immunity that causes rejection of the therapeutic protein. We hypothesized that rapamycin (RAPA) would diminish immunity due to vector-delivered recombinant dystrophin in the adult mdx mouse model for DMD. To test this hypothesis, we injected limb muscle of mdx mice with RAPA-containing, poly-lactic-co-glycolic acid (PLGA) microparticles prior to dystrophin gene transfer and analyzed treated tissue after 6 weeks. RAPA decreased host immunity against vector-mediated dystrophin protein, as demonstrated by decreased cellular infiltrates and decreased anti-dystrophin antibody production. The interpretation of the effect of RAPA on recombinant dystrophin expression was complex because of an effect of PLGA microparticles.National Institutes of Health (U.S.) (F31-NS056780-01A2)National Center for Research Resources (U.S.) (KL2 RR024154)United States. Army Medical Research and Materiel Command (grant W81XWH-05-1-0334

MSC Pretreatment for Improved Transplantation Viability Results in Improved Ventricular Function in Infarcted Hearts

Many clinical studies utilizing MSCs (mesenchymal stem cells, mesenchymal stromal cells, or multipotential stromal cells) are underway in multiple clinical settings; however, the ideal approach to prepare these cells in vitro and to deliver them to injury sites in vivo with maximal effectiveness remains a challenge. Here, pretreating MSCs with agents that block the apoptotic pathways were compared with untreated MSCs. The treatment effects were evaluated in the myocardial infarct setting following direct injection, and physiological parameters were examined at 4 weeks post-infarct in a rat permanent ligation model. The prosurvival treated MSCs were detected in the hearts in greater abundance at 1 week and 4 weeks than the untreated MSCs. The untreated MSCs improved ejection fraction in infarcted hearts from 61% to 77% and the prosurvival treated MSCs further improved ejection fraction to 83% of normal. The untreated MSCs improved fractional shortening in the infarcted heart from 52% to 68%, and the prosurvival treated MSCs further improved fractional shortening to 77% of normal. Further improvements in survival of the MSC dose seems possible. Thus, pretreating MSCs for improved in vivo survival has implications for MSC-based cardiac therapies and in other indications where improved cell survival may improve effectiveness

Schematic diagram of three-stage differentiation protocols.

<p>Both MenSCs and BMSCs were sequentially treated by three combinations of cytokines, growth factors, and hormones under commitment, differentiation and maturation steps.</p

Evaluation of multipotency and chromosomal stability of isolated MenSCs <i>versus</i> BMSCs.

<p>(A) MenSC and BMSCs differentiation into osteoblasts (ii), chondrocytes (iii) and adipocytes (iiii) judged by Alizarin red staining, immuostaining of Collagen type II and Oil red O staining, respectively; Scale bar: 100 µm. (B) Chromatograms illustrating no chromosomal aberrations in MenSCs at passage 12 compared to cells at passage 2. GeneMarker plots showing results of MLPA analysis. Green lines illustrated the upper and lower limits of acceptable ranges of variations in MLPA analysis. Green dots show the chromosomal locations which are balanced and the red dots in the upper side of the plots show chromosomal gain and red dots in lower side of the plots show chromosomal loss.</p

Functionality characteristics of differentiated MenSCs compared to BMSCs.

<p>(A) ALB levels (ng/ml/48 h) in cell supernatants at days 0, 10, 15, and 25 of differentiation. † indicates significant difference between specified day and the previous time period of differentiation in the same stem cell (<i>P<0.05</i>), ‡ indicates significant difference (<i>P<0.05</i>) between differentiated MenSCs and BMSCs at last day of differentiation. (B) PAS staining of glycogen storage in fetus liver and HepG2 as positive control, undifferentiated and differentiated MenSCs and BMSCs by various protocols (P1–P3). (C) Expression pattern of Cytochrome P450 7A1 (<i>CYP7A1</i>) in reference to GAPDH in differentiated MenSCs and BMSCs by various protocols. Undif: undifferentiated cells, W: water.</p

<i>In vivo</i> assay of tumorigenicity and immunological reaction of MenSCs.

<p>(A) 2×10⁶ cells were subcutaneously injected in the dorsolateral part of the flank of nude mice (i), No tumor formation was observed in treated mice (ii), The excised tissues were fixed in buffered formalin, embedded in paraffin, and sectioned in 5-µm sections (iii). (B) The sectioned tissues were evaluated using H & E staining. (C) Immunoreactivity of mice sera to cultured MenSCs was evaluated using immunofluorescence staining. The cells (2×10⁴ cells per slide) were fixed in acetone at −20°C for 5 min and were incubated for 1 hour at 4°C with mice sera. Subsequently, the cells were washed three times with PBS and incubated with FITC-labeled sheep anti-mouse IgG at RT for 45 min in the dark. DAPI was used for nuclear staining.</p

Morphology of MenSCs compared to BMSCs during differentiation by three protocols.

<p>The gradual change of MenSC morphology compared with BMSCs under each differentiation protocol (P1–P3) has been shown by phase contrast photographs. Scale bar: 100 µm.</p