The authors have declared that no competing interests exist.
Conceived and designed the experiments: SWH. Performed the experiments: PKMM RSS VYS CMT GBP FHdS VGP FLNM AHO. Analyzed the data: PKMM YMCSM RC VD SWH. Contributed reagents/materials/analysis tools: SWH YMCSM RC VD. Wrote the paper: SWH PKMM.
Mucopolysaccharidosis type I (MPSI) is an autosomal recessive disease that leads to systemic lysosomal storage, which is caused by the absence of α-L-iduronidase (IDUA). Enzyme replacement therapy is recognized as the best therapeutic option for MPSI; however, high titers of anti-IDUA antibody have frequently been observed. Due to the immunosuppressant properties of MSC, we hypothesized that MSC modified with the IDUA gene would be able to produce IDUA for a long period of time. Sleeping Beauty transposon vectors were used to modify MSC because these are basically less-immunogenic plasmids. For cell transplantation, 4×106 MSC-KO-IDUA cells (MSC from KO mice modified with IDUA) were injected into the peritoneum of KO-mice three times over intervals of more than one month. The total IDUA activities from MSC-KO-IDUA before cell transplantation were 9.6, 120 and 179 U for the first, second and third injections, respectively. Only after the second cell transplantation, more than one unit of IDUA activity was detected in the blood of 3 mice for 2 days. After the third cell transplantation, a high titer of anti-IDUA antibody was detected in all of the treated mice. Anti-IDUA antibody response was also detected in C57Bl/6 mice treated with MSC-WT-IDUA. The antibody titers were high and comparable to mice that were immunized by electroporation. MSC-transplanted mice had high levels of TNF-alpha and infiltrates in the renal glomeruli. The spreading of the transplanted MSC into the peritoneum of other organs was confirmed after injection of 111In-labeled MSC. In conclusion, the antibody response against IDUA could not be avoided by MSC. On the contrary, these cells worked as an adjuvant that favored IDUA immunization. Therefore, the humoral immunosuppressant property of MSC is questionable and indicates the danger of using MSC as a source for the production of exogenous proteins to treat monogenic diseases.
Mucopolysaccharidosis type I (MPSI) is an autosomal recessive disease that leads to systemic lysosomal storage caused by the absence of the enzyme alpha-L-iduronidase (IDUA)
Currently, with the high production capacity of the recombinant IDUA enzyme, enzyme replacement therapy (ERT) has become the best therapeutic option for MPSI. Although the cost of treatment is very expensive (US$ 150–300 thousand per year), patients treated weekly with this enzyme via intravenous infusion have shown great improvement. Dramatic reduction in urinary GAG excretion, normalization of hepatosplenomegaly and improved respiratory function and physical capacity were the main benefits that were observed in most patients treated by ERT
The IDUA in circulation is taken up by cells via mannose-6-phosphate receptor through a mechanism known as cross-correction. For efficient ERT, it is essential to maintain the active catalytic site of the enzyme and that these enzymes penetrate efficiently into deficient cells. Despite the existence of this transport mechanism for IDUA, most MPSI patient cells have never interacted with this enzyme. Therefore, IDUA becomes a foreign body that can generate an immune response. In clinical studies of lysosomal storage diseases (LSD) by ERT, alloantibodies were generated in all LSD
Mesenchymal stem cells (MSC) are able to differentiate into osteocytes, chondrocytes, adipocytes and other cells and are capable of proliferation and adhesion to plastic, which facilitates their cultivation and expansion in large quantities
The immunosuppressant effects of MSC upon T cells, natural killer cells, dendritic cells and macrophages have been widely studied
Because the antibody generation against IDUA is a serious problem when treating MPSI patients with ERT and based on the immunosuppressive property of MSCs, we hypothesized that MSCs modified with an IDUA gene can constitutively produce IDUA because the MSCs could decrease or avoid the generation of anti-IDUA antibodies. To test this hypothesis, we modified MSC with a Sleeping Beauty transposon (SB) vector expressing the human IDUA gene to constantly provide IDUA
In this study, we used the SB system for gene transfer because it is an integrative, non-viral vector and therefore it is expected to bring about long-term gene expression and an immune response against the vector should be minimal because this system is completely void of viral proteins, which can trigger undesired immune reactions.
All procedures involving animals were performed with the approval of the Research Ethics Committee of the Federal University of São Paulo, Brazil (Approval number: CEP 1278/07). IDUA knockout mice (KO)
The cDNA encoding the human IDUA gene was excised from the pTiger vector
The KO and WT mice were euthanized by cervical dislocation to obtain mesenchymal stem cells (MSC-KO and MSC-WT, respectively) by flushing the bone marrow of the femur and tibias. Technologies, Carlsbad, USA) supplemented with 10% fetal bovine serum (Life Technologies), 2 mM glutamine (Life Technologies), 50 units/ml penicillin and 50 mg/ml streptomycin sulfate (Life Technologies). The osteogenic and adipogenic differentiation were performed based on an established protocol
The KO and WT mice were treated with 80 mg/kg of isosorbide mononitrate by gavage two hours previous to the procedure. MSC-KO and MSC-WT were nucleofected with the pT2-CAGGS-IDUA and pCMV-SB100X vectors (MSC-KO-IDUA and MSC-WT-IDUA, respectively) and were expanded for 15 days. Four million cells were diluted in 4 ml of DMEM and were injected into the peritoneum of each mouse.
In vitro IDUA dosage was performed using a previously described protocol
The presence of human anti-IDUA antibody was determined using a method described by Di Domenico et al.
The tissues were fixed in 4% paraformaldehyde for 48 hours, dehydrated and embedded in paraffin. Sections of 4-μm thickness were obtained and stained with hematoxylin-eosin (HE) to determine the degree of tissue regeneration and the presence of adipocytes and infiltrated cells. Images were obtained using an optical microscope (Olympus BX60) and analyzed digitally
The MSC-KO was cultivated in the previously described conditions. Three million cells were incubated with 10 μCi of 111In-oxine for 30 minutes at 37°C. 111In-labeled MSC were injected intraperitoneally into six 3-month-old WT mice. Two, four and twenty-four hours after injection, these mice were euthanized by cervical dislocation and the tissues were collected and weighed. The 111In-oxine level was measured using the 1282 Compugamma program (LKB Wallac, Gaithersburg, Md.), and the radioactivity in each organ was expressed in two ways: by the counts per unit mass and as a percentage of the injected dose. In all cases, the radioactive decay of 111In was corrected to the time of injection. Differences in the radioactivity of the measured organs were determined using analysis of variances (ANOVA) at a threshold of p = 0.05 to indicate a statistical significance.
The GM-CSF, IFNγ, TNFα, IL-2, IL-4, IL-5, IL-10 and IL-12 in treated KO mice serum from MSC-KO-IDUA (n = 3) and non-treated KO mice (n = 2) were measured fifteen days after the third cell injection using a Bio-Plex Pro Mouse Cytokine 8-Plex panel (Bio-rad, Hercules, CA) in Luminex and analyzed using the Bio-Plex Manager 6.0 software (Bio-rad).
Using a 1 ml insulin syringe, 50 μg of plasmid DNA in 50 μl of PBS was delivered into each of the quadriceps muscles of the mice (25 μg pT2-CAGGS-IDUA plus 25 μg pCMV-SB100 or pCMV-ΔDDE per mouse). Immediately after the DNA injection, electroporation was performed using a needle electrode of 0.5 cm needles of 0.5 mm thickness and with a 5 mm distance between them. Three electric pulses (field strength = 100 V/cm; pulse length = 50 ms; ECM 830 field generator, BTX Division, Genetronix, San Diego, CA, USA) were delivered at 1 s of intervals
MSC were established according to the culture criteria that were described previously. The MSC-KO were maintained in culture for up to 40 passages without morphological changes or differentiation potentials in osteocytes and adipocytes (
MSC-KO cells were differentiated into osteoblasts (upper panel) and adipocytes (lower panel) before (A) and after nucleofection (B, C). Deposits of fat and calcium, which are characteristic of adipocytes and osteoblasts, respectively, are stained in yellow and red, respectively. The original magnification is 100×, and the bars correspond to 50 μm.
To obtain a large amount of IDUA-producing MSC, the MSC-KO were nucleofected with the following plasmids: pT2-CMVi-IDUA, pT2-IDUA and pT2-CAGGS-IDUA (
CMV: minimum human cytomegalovirus promoter; CMVi: complete human cytomegalovirus promoter; CAGGS: chicken β-actin promoter with CMV enhancer; IDUA: human IDUA cDNA; pA: polyadenylation signal; IR L: left inverted repeated sequence; IR D: right inverted repeated sequence; SB100X: Sleeping Beauty 100X; ΔDDE: Mutated Sleeping Beauty without transposase activity.
IDUA activity of all nucleofected cells were monitored for 30 days, except for the pT2-CAGGS-IDUA + pCMV-SB100X nucleofected cells, which were monitored for one year. *p<0.0001 for the pT2-CAGGS-IDUA+pCMV-SB100X group compared to other groups. A two-way ANOVA with the Bonferroni post hoc test was used. Vector descriptions are in the Methodology section.
To determine the biodistribution of the injected MSC, the MSC were radiolabeled with Indium-111 that was conjugated to oxine, injected into the peritoneum of the mice, and the organs were isolated for radioactivity counting. Two hours after MSC injection, radioactivity was detected in the spleen, stomach, large and small intestines, liver and kidney; and 24 hours later, the profile of radioactivity distribution was quite similar to that of 2 hours after injection (
MSC were labeled with 111In before injection, and radioactivities in the isolated organs were counted 2 (A) and 24 hours (B) later. Five and three mice were used for 2- and 24-hour experiments, respectively. * p<0.05 when comparing the spleen to all other tissues. A one-way ANOVA with the Bonferroni post hoc test was used.
For the first MSC-KO implantation, the cells were nucleofected with pT2-CAGGS-IDUA and pCMV-SB11, and 4×106 of these cells that were suspended in 4 ml were injected into the peritoneum of 4-month-old KO mice. These mice produced 28.8±58.7 U/mg of IDUA per mouse, and the final volumes and protein concentrations of the crude extracts were usually 1 ml and 3 mg/ml, respectively; therefore, at the moment of cell injection, these cells were producing approximately 9.6 U of IDUA. Taking into account that a mouse weighing 25 g contains approximately 2 ml of blood, the initial IDUA activity of the MSC-transplanted mouse should be approximately 4.8 U, which corresponds to the activity of a wild-type mouse
For the first transplantation (A), four million MSC-KO cells were nucleofected with pCMV-SB11 and pT2-CAGGS-IDUA and transplanted into the peritoneum. Three mice died during this experiment: 88.1.2, 88.1.5 and 89.1.3. Approximately, 40 days after the first cell transplantation, the second transplantation was performed using MSC-KO that were nucleofected with pCMV-SB100X and pT2-CAGGS-IDUA (B). Here, two new IDUA-KO mice were included (#). A month after the second transplantation, a third transplantation was performed using the same procedure as the second (C). Fifteen days after the last cell transplantation, blood samples were collected to quantify the anti-IDUA antibody (D). *p<0.05 when comparing the IDUA-KO group to other groups. A two-way ANOVA with the Bonferroni post hoc test was used. HT: heterozygous mouse. KO: IDUA-KO mouse.
One month after the first cell transplantation, these animals were treated again with 4×106 MSC that were modified with SB100X, which produced 359.22±108.16 U/mg. Based on the same calculation as before, we conclude that 120 U was injected into the peritoneum, and this value was 12-fold higher than what was used in the first transplantation. In this experiment, two new KO mice were included for comparison with the other ongoing mice. One day after cell transplantation, more than 1 unit of IDUA activity was present in the blood of the three mice, which represented about a half of the activity of heterozygous mice (
When these mice reached approximately 6-months-old, the third injection of MSC-KO-IDUA was carried out with the intention of reverting, at least partially, the disease progression. At this time, we injected the same number of cells, but they produced more IDUA activity: 530.66±59.72 U/mg, which represents an injection of 179 U. The six-month-old KO mice were used to be weakened because of disease progression; consequently, any treatment in this stage was a challenge. After a week of follow-up, we did not detect any IDUA activity in these mice (
To evaluate the antibody response that was generated against IDUA in the MSC-KO, the same cell transplantation procedure was performed using a MSC-WT that was modified with the same vectors to express IDUA. In this step, we used the MSC-WT to eliminate any interference from the IDUA mutation in the immunosuppressant property of MSC. The MSC-WT-IDUA produced 461.55±52.05 U/mg, which corresponded to 154 U before injection into the peritoneum. Therefore, these values were very similar of those that were used in the previous MSC-KO-IDUA transplantation. After two weeks of cell transplantation, anti-IDUA antibody was detected in all mice and was present until the last assay, which occurred on the 98th day (
MSC from wild-type mice (C57/Bl6) were nucleofected with pCMV-SB100X and pT2-CAGGS-IDUA and transplanted into the peritoneum of wild-type mice (n = 6 per group) following the same procedure that was used for the KO mice (A). In the negative control group, pT2-CAGGS-IDUA and pCMV-ΔDDE were used. For DNA immunization, pT2-CAGGS-IDUA with pCMV-SB100X or with pCMV-ΔDDE vectors were injected into the thighs of the mice and underwent electroporation (n = 5 per group). A two-way ANOVA with Bonferroni post hoc test was used.
To understand the immunogenicity of IDUA, WT mice were transfected with the same vectors by electroporation. Electroporation was adopted here because this method brought about better immunization
As the final experiments, the MSC- KO- IDUA treated mice were killed and their organs were analyzed by histology. Among the changes observed, we found inflammatory infiltrate in the renal glomeruli, thickening of the Bowman's capsule, reduction of the lumen of the renal tubules and replacement of normal tissue by inflammatory infiltrate in the renal medulla (
Kidneys from three treated mice were stained with Hematoxylin-Eosin (A). Inflammatory infiltrate in the cortex (*), replacement of normal tissue by inflammatory infiltrate in the renal medulla (→), thickening of the Bowman's capsule (???) and reduction of the lumen of the renal tubules (▹) were marked in the figure. Cytokine production was then evaluated using blood samples from the three treated and two non-treated mice 15 days after the last cell transplantation (B). Only TNF-alpha was detected in the treated mice. *p<0.0001 by the paired student t-test.
The cytokine profiles of MSC-KO-IDUA-treated mice were then analyzed using blood samples that were collected 15 days after the last cell transplantation, and the non-treated WT mice were used as a control. Among the analyzed cytokines, only TNF-alpha was present in MSC-KO-IDUA-treated mice and was 5-fold higher than that of the control group (
Monogenic diseases are of great interest for gene therapy studies because these diseases currently have no effective treatment. MPSI patients must have a constant supply of IDUA to relieve disease manifestation, and this can be performed by enzyme replacement therapy. It has been observed that anti-IDUA antibodies can be generated by ERT
The SB system has been proven to be efficient to gene transfer and long-term gene expression in many mammalian cells, including MSC
Because MPSI is a monogenic disease that affects all patient cells, an ideal therapy should be one that could provide IDUA to all affected cells, but this practice is not feasible at this moment. The peritoneum is a highly vascularized region that is easy to access; therefore, a large volume of MSC can be injected. These injected cells can rapidly traffic to affected organs, such as the liver, spleen and kidneys, because of its proximity to these organs. In addition, secreted IDUA will circulate easily and provide this enzyme to the body. The ability for MSC trafficking through the peritoneum is not clearly known, but because macrophages are located in this space and they can traffic through other organs
These data indicate that the distribution of the MSC-KO-IDUA through the body by intraperitoneal injection was an easy and productive procedure.
To test the hypothesis that MSC modified with IDUA could become a good, long-term source of IDUA in vivo because of the immunosuppressant properties of MSC, four million MSC-KO-IDUA were injected into the peritoneum three times over one-month intervals. In an attempt to minimize the immune response, MSC were modified with SB11 to produce a low level of IDUA and were used in the first injection, and for the second and third injections, they were modified with SB100X to produce high levels of IDUA. Considering that the total blood volume of a mouse weighing 25 g is approximately 2 ml, the expected IDUA activities in the blood after injections are 4.8, 60 and 90 U/ml for the first, second and third injections, respectively. Therefore, the units produced by the first injection are similar to that produced by a wild-type mouse (4±2.5 U/ml)
The immunosuppressant activity of MSC in vivo seems to be controversial. For example, the treatment of the autoimmune disease Systemic Lupus Erythematosus (SLE) with bone marrow MSC increased the survival and decreased the level of circulating anti-dsDNA
In conclusion, our in vivo study with MSC modified to constitutively produce IDUA showed an unexpected adjuvant effect of MSC for immunization, which raised high titers of an anti-IDUA antibody. This antibody response was as strong as DNA immunization by electroporation and lasted longer. Therefore, the use of genetically modified MSC for the long-term production of IDUA in KO mice to treat MPSI still faces unavoidable antibody responses. Our studies have been carried out in a murine model using the human IDUA gene, but the use of human MSC as a source for production of exogenous proteins to treat monogenic diseases must be well validated before it is clinically applied.