5. The optimal TGF-β1 inhibitor

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.

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

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.

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).


In order to demonstrate PMO inhibition, THP-1 cells, which are human monocytes that express TGF-β, were studied. THP-1 grows equally well in RPMI supplemented with 10% fetal bovine serum and serum-free media. When grown in serum-free media, TGF-β is not secreted into the media (ELISA = 0 pg./mL). Media supplemented with 50 ng/mL PMA lead to TGF-β secretion (ELISA = 92 pg./mL). The addition of 10 μM atorvastatin (Lipitor) enhances TGF-β

The evaluation of mRNA from splice altering PMOs is shown (Table 3). The control fragment appears at the correct size. Cells treated with PMOs targeting SD Ex2, SD Ex4, and AUG show no variation in transcript size and thus no evidence of exon skipping. The AUG signal appears to be enhanced relative to the untreated control, possibly indicating a rebound induction of transcription. This may be anticipated as the translation start site inhibitor will lead to suppression of the propeptide including LAP, which may lead to loss of the negative feedback mechanism for TGF-β1 transcription. Cells treated with PMOs targeting SD Ex5 and SD Ex6 reveal smaller transcripts in addition to faint bands at the correct size. The SD Ex5 smaller transcript is consistent in size with the loss of exon 4 (74 bp), which would leave the mature mRNA in frame. Those transcripts skipping exon 5 would be smaller yet (148 bp) and are expected to be degraded by a nonsense-mediated decay (NMD), so that the product would not be observed. The SD Ex6 smaller fragment is approximately 800 bp in size, which is 300 bp smaller than the expected full-length transcript and consistent with loss of both exons 5 (148 bp) and 6 (156 bp), which would be 304 bp smaller than the full-length transcript.

> Mu TGF-β1 protein (pg/mL) 5 μM 65 h

Functional Activation of Autologous Human Diabetic Stem Cells for Cell Therapy

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

Cell viability (% control)

secretion by fivefold (ELISA >500 pg./mL) following 72 h of incubation.

Hu TGF-β1 protein

SD refers to the splice donor site of the exon (Ex) and SA refers to the splice acceptor site.

Table 3. Exon skipping in THP-1 cells stimulated to secrete TGF-β1.

No PMO 520 2 — 100 Scr Ctr 502 15 1700 10 88 SD Ex1\* <sup>385</sup> <sup>7</sup> — — SA Ex2 387 5 — — SD Ex2 BLD 1320 30 93 SA Ex3 594 21 — — SD Ex3 222 3 — — SA Ex4 465 12 — — SD Ex4 23 2 520 20 66 SA Ex5 — — SD Ex5 BLD 120 10 79 SA Ex6 404 18 — — SD Ex6 BLD 330 10 79 SA Ex7 101 1 — 91

(pg/mL)

Treatment (5 μM 96 h)

\*

Table 2. Oligomer sequences employed to inhibit TGF-β1 translation.

diminished inhibition of translation. By contrast, the PMO with three inosines, TGF-β1 3-I (1067), inhibited translation more effectively than PMOs with all guanine or PMOs in which one or two guanines were replaced by inosine (data not shown).

TGF-β1 has seven exons transcribed into eight variant mRNAs, five alternately spliced variants and three unspliced forms (Figure 7). A small signal peptide (29 amino acids) is encoded in exon 1; the precursor LAP is encoded in exons 1 through 5; the active TGF-β1 is encoded in exons 6 and 7. The LTBP is encoded by a separate gene and binds directly to the LAP in the latent TGF-β complex prior to secretion. The amino terminus of LTBP binds to the extracellular matrix followed by proteolytic cleavage by a serine protease, plasmin, releasing the latent complex. A urokinase plasminogen activator (uPA) protease cuts the 391-amino acid TGF-β1 propeptide liberating the active 112 amino acid TGF-β1, which forms a homodimer ligand for the TGF-β receptors [47].

The TGF-β N terminal domain is present in a variety of proteins, which include TGF-β, decapentaplegic peptides, and bone morphogenetic proteins. The N-terminal domain expressed on the decapentaplegic protein acts as an extracellular morphogen guiding: (1) the proper development of the embryonic dorsal hypoderm, (2) viability of larvae, and (3) cell viability of the epithelial cells in the imaginal disks. When the N terminal domain is expressed on the bone morphogenetic protein (BMP), it induces cartilage and bone formation, possibly for epithelial osteogenesis. TGF-β1 is a protein composed of 112 amino acid residues liberated by proteolytic cleavage from the C-terminal of a precursor protein. A number of proteins are related to TGF-β1. The TGF-beta family is only active as homo- or heterodimers, the two chains being linked by a disulphide bond. X-ray studies of TGF-β2 reveal that all the other cysteines are involved in intrachain disulphide bonds. The four disulphide bonds in TGF-β and in the inhibin beta chains distinguish function from the other members of this family that lack the first bond. Concern has been noted as TGF-β not only exerts tumor-suppressive effects but also modulates cell invasion and immune regulation such that dysregulation of the TGF-β signaling pathway can result in tumor development.

In order to demonstrate PMO inhibition, THP-1 cells, which are human monocytes that express TGF-β, were studied. THP-1 grows equally well in RPMI supplemented with 10% fetal bovine serum and serum-free media. When grown in serum-free media, TGF-β is not secreted into the media (ELISA = 0 pg./mL). Media supplemented with 50 ng/mL PMA lead to TGF-β secretion (ELISA = 92 pg./mL). The addition of 10 μM atorvastatin (Lipitor) enhances TGF-β secretion by fivefold (ELISA >500 pg./mL) following 72 h of incubation.

The evaluation of mRNA from splice altering PMOs is shown (Table 3). The control fragment appears at the correct size. Cells treated with PMOs targeting SD Ex2, SD Ex4, and AUG show no variation in transcript size and thus no evidence of exon skipping. The AUG signal appears to be enhanced relative to the untreated control, possibly indicating a rebound induction of transcription. This may be anticipated as the translation start site inhibitor will lead to suppression of the propeptide including LAP, which may lead to loss of the negative feedback mechanism for TGF-β1 transcription. Cells treated with PMOs targeting SD Ex5 and SD Ex6 reveal smaller transcripts in addition to faint bands at the correct size. The SD Ex5 smaller transcript is consistent in size with the loss of exon 4 (74 bp), which would leave the mature mRNA in frame. Those transcripts skipping exon 5 would be smaller yet (148 bp) and are expected to be degraded by a nonsense-mediated decay (NMD), so that the product would not be observed. The SD Ex6 smaller fragment is approximately 800 bp in size, which is 300 bp smaller than the expected full-length transcript and consistent with loss of both exons 5 (148 bp) and 6 (156 bp), which would be 304 bp smaller than the full-length transcript.


\* SD refers to the splice donor site of the exon (Ex) and SA refers to the splice acceptor site.

Table 3. Exon skipping in THP-1 cells stimulated to secrete TGF-β1.

diminished inhibition of translation. By contrast, the PMO with three inosines, TGF-β1 3-I (1067), inhibited translation more effectively than PMOs with all guanine or PMOs in which

Name Sequence 5<sup>0</sup> ! 3<sup>0</sup> Mol Wt. GT Control CCTCCTACCTCAGTTACAATTTATA — 144 Control AGTCTCGACTTGCTACCTCA 7020 TGF-β1 GT GCACTGCCGAGAGCGCGAACA 7642 TGF-β1 GAGGGCGGCATGGGGGAGGC 7175 TGF-β1 1-I\* GAGGGCGGCATGG I GGAGGC 7160 TGF-β1 3-I (1067) GAGGGCGGCATG III GAGGC 7130 TGF-β1 2-I GAG I GCGGCATGG I GGAGGC 7145 TGF-β1 4-I GAG I GCGGCATG III GAGGC 7115

TGF-β1 has seven exons transcribed into eight variant mRNAs, five alternately spliced variants and three unspliced forms (Figure 7). A small signal peptide (29 amino acids) is encoded in exon 1; the precursor LAP is encoded in exons 1 through 5; the active TGF-β1 is encoded in exons 6 and 7. The LTBP is encoded by a separate gene and binds directly to the LAP in the latent TGF-β complex prior to secretion. The amino terminus of LTBP binds to the extracellular matrix followed by proteolytic cleavage by a serine protease, plasmin, releasing the latent complex. A urokinase plasminogen activator (uPA) protease cuts the 391-amino acid TGF-β1 propeptide liberating the active 112 amino acid TGF-β1, which forms a homodimer ligand for

The TGF-β N terminal domain is present in a variety of proteins, which include TGF-β, decapentaplegic peptides, and bone morphogenetic proteins. The N-terminal domain expressed on the decapentaplegic protein acts as an extracellular morphogen guiding: (1) the proper development of the embryonic dorsal hypoderm, (2) viability of larvae, and (3) cell viability of the epithelial cells in the imaginal disks. When the N terminal domain is expressed on the bone morphogenetic protein (BMP), it induces cartilage and bone formation, possibly for epithelial osteogenesis. TGF-β1 is a protein composed of 112 amino acid residues liberated by proteolytic cleavage from the C-terminal of a precursor protein. A number of proteins are related to TGF-β1. The TGF-beta family is only active as homo- or heterodimers, the two chains being linked by a disulphide bond. X-ray studies of TGF-β2 reveal that all the other cysteines are involved in intrachain disulphide bonds. The four disulphide bonds in TGF-β and in the inhibin beta chains distinguish function from the other members of this family that lack the first bond. Concern has been noted as TGF-β not only exerts tumor-suppressive effects but also modulates cell invasion and immune regulation such that dysregulation of the TGF-β signal-

one or two guanines were replaced by inosine (data not shown).

Table 2. Oligomer sequences employed to inhibit TGF-β1 translation.

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

I refers to inosine, a strategy to limit "purine clash."

the TGF-β receptors [47].

\*

ing pathway can result in tumor development.

Skipping exon 6 alone or exon 5 alone would be degraded by NMD, and those transcripts would not be observed. Skipping exons 5 and 6 will also remain in frame.

cell homing to SDF-1 ligand, (3) increases nitric oxide (NO) release, stimulating stem cell migration, and (4) increases vascular repair by the activated diabetic CD34+ cells. The transient TGF-β1 inhibition approach holds potential to impact other diabetic microvascular complications and improve current bone marrow transplantation processes used in the treatment of blood cancers. The transient inhibition of TGF-β1 in autologous diabetic CD34+ cells with an antisense PMO ex vivo represents a feasible approach that poses minimal potential for adverse events and has potential benefit to the patient with diabetic retinopathy. Challenges remain in development such as the selection of animal models that adequately predict the human response to treatment. Numerous features including the genetic basis of the retinal disease, anatomical differences in the eye, and the genesis of retinal damage limit the utility of animal models. Substantial differences in diabetic subpopulations, the presence of comorbidities, patient age, and diabetes severity will influence the success of our proposed therapy. A detailed understanding of the natural history of diabetic retinopathy deserves in-depth investigation, so that patient enrollment can be refined, and clinical trials will examine optimal patient populations and appropriate stage of disease. Current efforts are ongoing to address these limitations as

Functional Activation of Autologous Human Diabetic Stem Cells for Cell Therapy

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

Damaged retinal vessels are repaired by HSC in individuals throughout their life. Diabetic HSC function is impaired, leading to the development of numerous clinically important conditions including diabetic retinopathy. Selective ex vivo manipulation of TGF-β1 in diabetic HSC represents a therapeutic approach to maintain, enhance, and restore vascular viability in the retina. The PMO offers transcript selective binding and transient interference with translation of TGF-β1. The PMO offers a feasible technology in which they enter HSC, can inhibit autocrine TGF-β1 signaling in HSC, and have an excellent safety profile. We presented the process of selecting TGF-β1 as an optimal transcript and the optimal PMO sequence targeting TGF-β1 mRNA. Our studies identified a transient interference with the translation of TGF-β1 in diabetic CD34+ HSC with an antisense PMO that will (1) upregulate the expression of CXCR4, enabling stem cell homing and adhesion to sites of vascular injury in the retina, (2) stimulation of nitric oxide production, enabling stem cell mobility, and (3) the release of cell cycle checkpoints, enabling stem cell proliferation and differentiation required for the repair of vascular lesions. The manipulated stem cell treatment strategy is making the transition from

We acknowledge the exacting experimentation performed by Dr. Ewa Sitnicka and Carl Storey for the murine stem cell/transplantation studies. We also thank Valerie Stepps and Casey Renée Haynes for their persistent, successful efforts, exploring the uptake of FITC-PMO by

our protocol advances to the clinic.

discovery to preparation for clinical evaluation.

Acknowledgements

7. Conclusions

The exercise to identify an optimal inhibitor of TGF-β1 involved screening multiple gene targets and dozens of PMO inhibitors. Qualitative differences between splice-altering strategies and translation inhibitors involve the preservation of feedback inhibition of the promoter. Translation inhibitors and splice-altering targets that induce a nonsense-mediated decay (NMD) prevent the synthesis of the negative feedback, resulting in compensatory transcription followed by rebound translation of TGF-β1. By contrast, skipping of exons 5 and 6 leads to translation products with altered function but includes the LAP portion of the translated product, resulting in a prolonged inhibition of TGF-β1. Transient inhibition of TGF-β1 is desired [48], so the optimal approach favors the AUG and NMD PMO over exon skipping and ligand-neutralizing antibodies. Translation inhibition is preferred over NMD because NMD responses may be less reliable.

#### 6. Stem cell therapy for diabetic retinopathy

The Centers for Disease Control and Prevention report that 4.2 million (28.5%) of US diabetics aged ≥40 years have diabetic retinopathy (DR) or damage to the small blood vessels in the retina that may result in loss of vision [49]. The direct costs for DR in the US were over \$4.5 billion, and the indirect economic impact was an additional \$5 billion. Retinopathy occurs in almost all patients with type 1 diabetes and 75% of patients with type 2 diabetes within 15 years of the manifestation of diabetes [50]. Over 12,000 diabetic patients become blind each year due to ocular complications [51]. Current therapy addresses the end stages of DR including laser photocoagulation, intravitreal antivascular endothelial growth factor (VEGF) agents such as Bevacizumab and Aflibercept, intravitreal corticosteroids such as Triamcinolone, and vitreoretinal surgery. CD34+ stem cells from diabetic patients cannot generate endothelial cells to repair the vasculature, instead generating more inflammatory monocytes [52]. The CD34+ stem cell therapy described here exploits the ability of these cells to differentiate into a wide variety of cell types to stimulate both vascular and neural regeneration to treat early stages of DR.

CD34+ cells are capable of homing to vascular lesions in the eye, mediating vascular repair [53]. The use of autologous CD34+ cells eliminates the significant complication of transplant rejection. However, diabetic CD34+ cells are dysfunctional, contributing to the diabetic complication of DR [54]. While CD34+ cells from healthy subjects could repair retinal capillaries in streptozotocin-induced diabetic mice, spontaneously diabetic obese BBZDR/Wor rats and neonatal mouse oxygen-induced retinopathy animal models CD34+ cells from diabetic mice could not [55]. The approach described here restores function to dysfunctional diabetic CD34+ cells.

TGF-β1 is overexpressed and may cause dysfunction in diabetic CD34+ cells, and correction of this overexpression can restore the regenerative ability of those cells in diabetics. TGF-β1 is the major regulator of the balance between CD34+ proliferation, differentiation, and quiescence. Transient inhibition of TGF-β1 with an optimal PMO (1) activates human CD34+ proliferation, whereas ID11 antibody does not, (2) enhances CXCR4 cell surface expression and effective stem cell homing to SDF-1 ligand, (3) increases nitric oxide (NO) release, stimulating stem cell migration, and (4) increases vascular repair by the activated diabetic CD34+ cells. The transient TGF-β1 inhibition approach holds potential to impact other diabetic microvascular complications and improve current bone marrow transplantation processes used in the treatment of blood cancers.

The transient inhibition of TGF-β1 in autologous diabetic CD34+ cells with an antisense PMO ex vivo represents a feasible approach that poses minimal potential for adverse events and has potential benefit to the patient with diabetic retinopathy. Challenges remain in development such as the selection of animal models that adequately predict the human response to treatment. Numerous features including the genetic basis of the retinal disease, anatomical differences in the eye, and the genesis of retinal damage limit the utility of animal models. Substantial differences in diabetic subpopulations, the presence of comorbidities, patient age, and diabetes severity will influence the success of our proposed therapy. A detailed understanding of the natural history of diabetic retinopathy deserves in-depth investigation, so that patient enrollment can be refined, and clinical trials will examine optimal patient populations and appropriate stage of disease. Current efforts are ongoing to address these limitations as our protocol advances to the clinic.
