4. TGF-β is an optimal stem cell target for CD34+ stem cells

An inhibitor of c-myc was identified based on antiproliferative effects in differentiated cells that block translation as well as create a dominant-negative variant of c-myc [40]. This inhibitor was effective in preventing coronary restenosis [41], preventing cyst growth in kidneys of polycystic kidney disease models [42], and reducing tumor growth [43]. We investigated the cmyc inhibitor in c-kit+/sca-1+ cells incubated with IL-3, IL-6, and SCF to drive cell proliferation of the stem cells. Incubation of these cells with saline was associated with a cell doubling halflife of 2.66 days, a scrambled sequence PMO (5'-GCTATTACCTTAACCCAG-3<sup>0</sup> ) had a doubling half-life of 2.554 days, and the c-myc inhibitor (5'-ACGTTGAGGGGCATCGTCGC-3<sup>0</sup> ) had a doubling half-life of 2.53 days. Unlike differentiated cells, the stem cells show no difference in cell proliferation when c-myc was inhibited. While inhibiting, c-myc did not influence proliferation rate; however, it did enhance stem cell differentiation as high proliferation potential (HPP) colony forming counts (CFC) rose from 3.8 HPP CFC in controls to 8.0 HPP-CFC in c-myc-inhibited cultures. This surprising observation suggested that c-myc inhibition stimulates stem cell differentiation and regulates self-renewal inspired studies to look at upstream signaling pathways in these stem cells. We studied the inhibition of ecotropic virus insertion-1 (EVI-1), which inserts in the DNA of murine stem cells and c-Kit, a stem cell marker along with c-myc and found that PMO-antisense treatment in vitro decreased LTR-HSC repopulating ability (Figure 4). Furthermore, the intra-peritoneal administration of PMO antic-myc reduces HSC-repopulating ability in vivo (Figure 5). These results represent an excellent functional control for PMO-TGF-β1 since these PMO antisense treatments do not promote HSC engraftment while PMO-TGF-β1 does.

efflux out of the cell, and tissue half-life will quickly lead to undetectable PMO and a transient inhibition of TGF-β in the stem cells. The overall exposure of PMO will be below 100,000 times the reported no observed adverse effect level (NOAEL) for a similar PMO in GLP toxicology studies [38, 39]. The use of PMO-treated CD34+ stem cells to treat patients with diabetic

3. Optimal TGF-β PMO inhibitors in human Lin-CD34+ CD45+ HSC

TGF-β is a family of multifunctional peptide cytokines with the capacity to regulate proliferation, differentiation, adhesion, migration, and other functions in many cell types. TGF-β receptors are found on most cells, and their signal transduction positively and negatively regulates many other growth factors. Secreted TGF-β is cleaved into a latency-associated peptide (LAP) and a mature TGF-β1 protein. TGF-β is latent in the form of a TGF-β1 homodimer, a LAP homodimer, and a latent TGF-β1-binding protein (LTBP). However, TGF-β1 homodimer can be active, and the mature protein may also form heterodimers with

The HSC is pluripotent immature cell that can generate daughter cells committed to all nine types of mature blood cells, including trillions of white blood cells, red blood cells, and platelets. HSCs are found in the bone marrow and also circulate in the peripheral blood. HSC possesses two key properties: (1) the ability to self-renew (generating HSC replicates) and (2) the ability to generate daughter cells that differentiate into fully functional blood cells (namely, asymmetrical HSC division in which one daughter cell remains a HSC and the other daughter

When the most primitive HSCs self-replicate, they produce daughter cells with a long (possibly unlimited) clonal life span. When HSC replication leads to differentiation divisions, they lose their multi-lineage potential and the corresponding lineage commitment accompanied by a progressive reduction in clonal life span. Previous studies have shown that ex vivo proliferation of HSC favors differentiation divisions at the expense of self-replication, resulting in a

An inhibitor of c-myc was identified based on antiproliferative effects in differentiated cells that block translation as well as create a dominant-negative variant of c-myc [40]. This inhibitor was effective in preventing coronary restenosis [41], preventing cyst growth in kidneys of polycystic kidney disease models [42], and reducing tumor growth [43]. We investigated the cmyc inhibitor in c-kit+/sca-1+ cells incubated with IL-3, IL-6, and SCF to drive cell proliferation of the stem cells. Incubation of these cells with saline was associated with a cell doubling half-

bling half-life of 2.554 days, and the c-myc inhibitor (5'-ACGTTGAGGGGCATCGTCGC-3<sup>0</sup>

) had a dou-

)

4. TGF-β is an optimal stem cell target for CD34+ stem cells

life of 2.66 days, a scrambled sequence PMO (5'-GCTATTACCTTAACCCAG-3<sup>0</sup>

retinopathy is expected to be safe and feasible.

74 In Vivo and Ex Vivo Gene Therapy for Inherited and Non-Inherited Disorders

other TGF-β family members.

cells are destined to mature).

complete loss of HSC.

ID11, a neutralizing monoclonal antibody to three isoforms of TGF- β (TGF- β1, 2, 3), added to stem cell cultures can replace growth factors and prevent apoptosis in mouse HSC [7]. Adding 100 c-kit+/sca-1+ cells to 96-well plates with no IL-3, IL-6, or SCF led to no cells observed after 5 days in culture. Adding ID11 to those cultures led to 37 � 7 cells, confirming that the antibody could replace growth factors. Further, the addition of the PMO targeting c-myc led to 10 � 3 cells at 5 days in culture, leading us to conclude that c-myc expression is required for the loss of TGF-β phenotype. It became apparent that ID11 effectively blocks extracellular TGF-β, but a PMO (5'-GCA CTG CCG AGA GCG CGA ACA-3<sup>0</sup> ) inhibitor of TGF-β translation could have the advantage of blocking autocrine signaling. Inhibiting TGF-β with either antibody or antisense PMO enhances HPP-CFC from progenitor cells [7, 20, 21] and can enhance hematopoietic reconstitution following bone marrow transplantation [6, 44, 45]. Importantly,

Figure 4. PMO targeting of c-myc, c-kit, and EVI-1 in ex vivo cultures of highly purified murine LTR-HSC. LTR-HSCs were isolated as previously described, then 25 cells per well were incubated for 5 days with PMO and hematopoietic growth factors followed by intravenous transplant into lethally (950 rads) irradiated mice. CD45.2 congenic LTR-HSCs were transplanted into CD45.1 recipients, so that donor LTR-HSC could be detected by monoclonal antibodies. Significantly fewer (p < 0.05) LTR-HSCs were observed in cultures treated with c-kit, EVI-1, and c-myc PMO compared to control, c-myc scramble, and c-kit scramble PMO after 3 months post-transplant.

the transplantation of Tfgβ1�/� bone marrow into lethally radiated TGF-β1+/+ recipients reconstitutes all hematopoietic lineages [46]. Taken together, these studies encouraged further exam-

We investigated PMO inhibitors of TGF-β receptor I (5'CAT GGT CCC TGC AGA GAG GA-3<sup>0</sup>

confirming that the signal transduction pathway is responsible for the phenotype, while blocking other pathways did not (Figure 6). We focused on the TGF-β1 ligand due to the short

We investigated the use of an antisense PMO targeting the AUG translation start site for efficacy in inhibiting TGF-β1 expression by hybrid arrest of translation. One possible outcome of a PMO at AUG1 will be for translation slippage to a translation initiation start site at amino acid 38, AUG38 (Figure 7). The resulting protein will not have the signal peptide, leading to the loss of appropriate subcellular localization, altered autocrine regulation, and possibly a protein with a shorter half-life. The diminished protein product fails to provide a negative feedback to the promoter, so enhanced transcription is expected. To test this hypothesis, we evaluated six oligomers targeting translation and two scrambled control sequences (Table 2). The compounds were evaluated in an in vitro translation assay using rabbit reticulocyte lysate and a luciferase fusion transcript with TGF-β1 mRNA. Each of the antisense PMOs effectively inhibited translation, and the scrambled control oligomers did not inhibit, confirming PMO sequence specificity. The TGF-β1 PMO included 13 guanines (G) in the 20-mer and presented water solubility limitations and reduced synthetic yield concerns. Replacing guanine with inosine improved both water solubility and synthetic yield. However, inosine pairing with cytosine involves two hydrogen bonds in contrast to the three hydrogen bonds between guanine and cytosine. The hypothesis is that the more inosine replacement of guanine in the oligomer will result in a lower binding energy between PMO and target RNA and subsequent

Figure 7. Optimal antisense strategy. Multiple PMOs were developed to inhibit translation initiation at the AUG site as well as targeting each exon at both splice donor and splice acceptor sites (black bars). Skipping exons 2, 3, 5, and 6 results

in out-of-frame reading, and a nonsense-mediated decay (NMD) of the transcript is expected (X circles).

) also led to the TGF-β-inhibited phenotype in CD34+ cells,

Functional Activation of Autologous Human Diabetic Stem Cells for Cell Therapy

and TGF-β receptor II (5'-GAC CCA TGG CAG CCC CCG TCG-3<sup>0</sup>

type to the TGF-β1 ligand inhibitor. Subsequent studies targeting SMAD 4 (5<sup>0</sup>

half-life, enabling rapid onset and transient inhibition properties of the treatment.

)

) to reveal the same pheno-

http://dx.doi.org/10.5772/intechopen.79650


ination of the TGF-β1-signaling pathway.

5. The optimal TGF-β1 inhibitor

CAT CCT TCA CCA TCA T-3<sup>0</sup>

Figure 5. In vivo activity of PMO targeting c-myc in short-term and long-term hematopoietic stem cells. Mice were treated with c-myc-PMO in vivo (intraperitoneal injection) for 2 or 11 days. At each time point, mice were sacrificed, and the femoral marrow was assayed for HSC levels using a murine transplantation model. Significant reductions in repopulating HSC (p < 0.05) were observed in mice treated with c-myc PMO compared to normal bone marrow.

Figure 6. Targeting stem cell pathways. Studies were conducted targeting c-myc, SMAD4, EVI-1, c-kit, TGF-βRI, TGFβRII, and TGF-β1 with PMOs designed to inhibit expression in HSC. Regulation of transcription factors by TGF-β1 is linked to stem cell homing through CXCR4 interaction with SDF-1 and release of nitric oxide and elevated migration. The MAP kinase pathway signaling reveals the potential mechanism for the prevention of apoptosis with TGF-β1 inhibition. The upstream regulation of c-myc and p53 by TGF-β1 inhibition allows stem cells to proliferate. Inhibition of TGF-β1 is the optimal target resulting in stem cell proliferation, homing, and migration of all favorable properties for autologous transplantation.

the transplantation of Tfgβ1�/� bone marrow into lethally radiated TGF-β1+/+ recipients reconstitutes all hematopoietic lineages [46]. Taken together, these studies encouraged further examination of the TGF-β1-signaling pathway.

We investigated PMO inhibitors of TGF-β receptor I (5'CAT GGT CCC TGC AGA GAG GA-3<sup>0</sup> ) and TGF-β receptor II (5'-GAC CCA TGG CAG CCC CCG TCG-3<sup>0</sup> ) to reveal the same phenotype to the TGF-β1 ligand inhibitor. Subsequent studies targeting SMAD 4 (5<sup>0</sup> -AAT CAT ACT CAT CCT TCA CCA TCA T-3<sup>0</sup> ) also led to the TGF-β-inhibited phenotype in CD34+ cells, confirming that the signal transduction pathway is responsible for the phenotype, while blocking other pathways did not (Figure 6). We focused on the TGF-β1 ligand due to the short half-life, enabling rapid onset and transient inhibition properties of the treatment.
