2. Feasibility: PMO entry into CD34+ stem cells

Evidence for receptor-mediated internalization of DNA came from studies with leukocytes over 30 years ago [22]. Uptake involves endocytosis based on chloroquine, a lysosomotropism agent, enhanced intracellular fluorescence, sodium azide inhibited internalization, and a punctate pattern observed in the cytoplasm [23]. Scavenger receptors on rat liver endothelial cells participate in uptake and play a prominent role in plasma clearance [24]. Many oligonucleotide uptake pathways have been described, but the adaptor protein AP2 M1 is involved in phosphorothioate oligonucleotide (PSO) uptake. siRNA targeting clathrin and caveolin had no effect on antisense activity but did decrease the uptake of fluorescently labeled oligonucleotides, highlighting multiple subcellular compartments that accumulate oligonucleotides but not all are associated with antisense activity. Abasic oligomers, backbone and sugar without nucleobase oligomers, were not transported into cells by the AP2 M1 pathway [25], pointing to the nucleobase as a recognition site for uptake. The neutral charge of PMO compounds sets them apart from ionic forms like PSO.

co-express transforming growth factor-β (TGF-β) type I and type II receptors and one or more of the three isoforms of the TGF-β ligand as a latent complex [2]. The three TGF-β ligands qualitatively and quantitatively differ in the responses they elicit with TGF-β1, a multifunctional regulator of hematopoietic progenitors in vivo and in vitro, depending on cell differentiation, growth factors, ligand concentration, and cell–cell contacts [3]. Autocrine signaling by TGF-β1 plays a critical role in lineage-specific reconstitution [4] and enables non-canonical signaling involving mTOR, Ras, MAPK, PI3K, AKT, RhoA, and JNK [5]. TGF-β is an important part of the stromal microenvironment that regulates several niche cells, which in turn regulate HSC. Selective manipulation of endogenous TGF-β1 in HSC represents a therapeutic approach to

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

transplantation to maintain, enhance, and restore tissue viability and organ function.

transplanted mice after LTR-HSCs were treated with an antisense TGF-β1 PMO [6].

and least risk to the modulation of gene expression in HSC.

2. Feasibility: PMO entry into CD34+ stem cells

of PMO essential to transient inhibition of TGF-β1.

Phosphorodiamidate morpholino oligomers (PMOs) resist degradation [11], enhance specificity through a no-pucker six-membered morpholine ring in place of the five-membered ribose or deoxyribose [12], and are net charge neutral with one non-bridging oxygen substituted with a dimethylamine residue [13]. PMOs binds to the target RNA, forming a PMO:RNA heteroduplex that can inhibit translation [14] or pre-mRNA splicing [15]. The cellular internalization of PMOs in different cell types is mixed and not robust unless entry is assisted by cell uptake technologies [16]. Given the impressive safety profile for unmodified PMO [17–19], conjugation with delivery enhancements adds risk. An unmodified PMO represents the most specific

Antisense TGF-β will reverse HSC growth arrest induced by TGF-β ligand, informing the reversibility of the ligand [20]. The antisense approach gains access to autocrine RNA expression over neutralizing antibodies, which targets protein. Transient antisense inhibition of autocrine TGF-β1 in HSC triggers a cascade of hematopoietic proliferation and differentiation that paracrine TGF-β cannot alter [21]. We report the kinetics of HSC internalization and efflux

Evidence for receptor-mediated internalization of DNA came from studies with leukocytes over 30 years ago [22]. Uptake involves endocytosis based on chloroquine, a lysosomotropism

Transient inhibition of TGF-β1 in HSC accelerates the engraftment of long-term repopulating HSC (LTR-HSC), permits successful transplantation with as few as 60 LTR-HSCs to rescue mice from lethal irradiation, and promotes the survival of LTR-HSC in the absence of growth factors [6]. This permits LTR-HSC transplant without cell expansion ex vivo prior to transplant. TGF-β1 regulates LTR-HSC entry into the cell cycle at G0 [7]. Conditional knock-out of the TGF-β1 type II receptor in adult mice has increased stem cell cycling and reduced transplantation ability [8]; likewise, the inhibition of TGF-β in normal HSC with neutralizing antibodies releases cells into the cell cycle [9]. Inhibiting SMAD 4 signaling, key to TGF-β signaling, decreased HSC self-renewal in vivo [10]. The rapid generation of donor neutrophils that are the last cells to regenerate in bone marrow transplantation (BMT) is observed in

We explored techniques to deliver PMO into cells in culture to improve bioavailability and efficacy including scrape loading [26], syringe loading [27], microinjection [28], osmotic loading [29], and complexation with cationic lipids [30]. These techniques suffer from limited efficiency and poor reproducibility and often leave residual biologically active carrier molecules in the culture media. We then explored a variety of cationic peptides conjugated to the PMO for an enhanced delivery including HIV-TAT [31] and a broad spectrum of arginine-rich peptides [32–34]. At present, the optimal delivery peptide is still composed of multiple arginines [35]. A concern for loading arginine into a stem cell was the role arginine plays in generating NO, a complication in interpreting observations of CD34+ activation. Thus, we examined unassisted entry in stem cells.

Earlier studies evaluating unassisted PMO entry into cultured cells revealed that primary cell cultures are more efficient in uptake than established cell lines. Uptake is independent of PMO sequence or the position of FITC conjugation (5<sup>0</sup> vs. 3<sup>0</sup> ends are equivalent) (Figure 1A) but dependent on concentration (Figure 1B), time, and temperature. There is a direct relationship between fluorescence intensity of CD34+ cells and PMO concentration. Localization to both cytoplasmic and nuclear compartments is observed, so that both pre-mRNA and mRNA targets are feasible. Uptake into hematopoietic lineages reveals that monocytes and dendritic cell uptake are efficient, while entry into CD8+ T-cells, CD4+ T-cells, and B-cells is minimal [36]. Viral infection activates some T-cell populations, resulting in permissive PMO uptake [37]. Current understanding of mechanisms involved in activation associated with PMO uptake is limited.

The first evidence of unassisted PMO entry into CD34+ cells came from microscopic observation of cells in which the visible uptake of FITC-PMO came within 15 min. Stem cells are unique in permissive unassisted PMO uptake. The maximal saturation of PMO uptake into HSC occurs within 2 h (Figure 2). Optimal uptake in terms of activation of HSC occurs after 16 h of FITC-PMO incubation. Stem cell positivity after 16 h was observed at 37�C with 70 � 12% FITC-positive CD34+ cells (n = 6), room temperature incubation led to 56 � 8% positive cells (n = 5), and 4�C incubation led to 30 � 19% positive cells (n = 6). The percent FITC-PMO-positive CD34+ cells were mirrored by cellular fluorescence defined by mean channel fluorescence with 112 � 47 at 37�C, 56 � 22 at room temperature, and 31 � 24 at 4�C incubation. Negative controls included CD34+ cells incubated with no FITC-PMO at 4�C, RT, and 37�C (n = 6) for each group, and no FITC-PMO-positive cells were observed.

PMO uptake was found to be time and dose dependent. Uptake reached 100% FITC-PMOpositive CD34+ cells between 1 and 6 h of incubation at 37C. Uptake determined as percentpositive CD34+ cells measured over time were linear (r2 0.94–0.94) (Table 1). Comparison of CD34+ cells recovered from diabetic individuals to non-diabetic individuals reveals that uptake is three to five times more rapid in non-diabetic CD34+ cells compared to those from diabetic individuals (Table 1, Figure 3). No loss in cell viability has been observed in protocols involving a 6-h incubation sufficient for 100% PMO-positive cells. Preliminary data suggest that treated stem cells will carry less than 5 μg PMO into the eye as a result of a combination of

<sup>c</sup> Ratios (expected) Time to 100% pos. Saturation ratio

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)

Norm<sup>a</sup> <sup>40</sup> <sup>μ</sup>g (N = 2) 17.6 1.1 (0.95) 3.3 (N/D 40) 6 h 3.0 (D/N 40) Diab<sup>b</sup> <sup>40</sup> <sup>μ</sup>g (N = 1) 5.3 5.2 (0.95) 3.4 (D 160/40) [4] 18 h 3.6 (D 40/160) [4] Norm 160 μg (N = 2) 98.0 7.3 (0.94) 5.6 (N 160/40) [4] 1 h 6.0 (N 40/160) [4] Diab 160 μg (N = 1) 18.1 8.2 (0.94) 5.4 (N/D 160) 5 h 5.0 (D/N 160)

Figure 3. The percent of blood-derived CD34+ cell taking up FITC-PMO. A. Percent-positive (black bar) and mean channel fluorescence (gray bar) CD34+ cells from healthy donors. B. Percent-positive (black bar) and mean channel fluorescence (gray bar) CD34+ cells from diabetic subjects. Uptake is not different in healthy and diabetic subjects, but the rates of maximum saturation of FITC-PMO (measure by MEAN CHANNEL FLUORECENCE) are delayed in diabetic

CD34+ cells relative to non-diabetic cells. CD34+ cells were pre-enriched and FACS sorted as described above.

Treatment group Rate percent pos./H (r<sup>2</sup>

CD34+ cells recovered from normal donors.

CD34+ cells recovered from diabetic donors.

Table 1. PMO uptake kinetics in CD34+ cells.

Correlation coefficient from linear regression analysis.

a

b

c

Figure 1. Concentration-dependent uptake of FITC-PMO into CD34+ human stem cells. A. Percent-positive CD34+ HSC on the ordinate and PMO concentration on the abscissa. B. Mean channel fluorescence of CD34+ HSC on the ordinate and PMO concentration on the abscissa. The uptake of PMOs is not sequence specific: 20-mer PMOs have similar entry kinetics, and percent-positive CD34+ cells are directly proportional to the PMO concentration in the medium. CD34+ cells were isolated from the blood of healthy subjects by pre-enriching the CD34+ by a lineage negative selection followed by FACS sorting of CD34+ CD45+ cells.

Figure 2. Time-dependent uptake of FITC-PMO into CD34+ human stem cells. After 3 h at 37C at 150 μg/mL 144-F (a FITC-control PMO), >95% of CD34+ cells became FITC-labeled. At 16 h in culture, the degree of FITC-144-F PMO was the same as the 3-h point. CD34+ cells were isolated and FACS sorted from the blood of healthy subjects as described in Figure 1.

PMO uptake was found to be time and dose dependent. Uptake reached 100% FITC-PMOpositive CD34+ cells between 1 and 6 h of incubation at 37C. Uptake determined as percentpositive CD34+ cells measured over time were linear (r2 0.94–0.94) (Table 1). Comparison of CD34+ cells recovered from diabetic individuals to non-diabetic individuals reveals that uptake is three to five times more rapid in non-diabetic CD34+ cells compared to those from diabetic individuals (Table 1, Figure 3). No loss in cell viability has been observed in protocols involving a 6-h incubation sufficient for 100% PMO-positive cells. Preliminary data suggest that treated stem cells will carry less than 5 μg PMO into the eye as a result of a combination of


a CD34+ cells recovered from normal donors.

b CD34+ cells recovered from diabetic donors.

c Correlation coefficient from linear regression analysis.

Table 1. PMO uptake kinetics in CD34+ cells.

Figure 1. Concentration-dependent uptake of FITC-PMO into CD34+ human stem cells. A. Percent-positive CD34+ HSC on the ordinate and PMO concentration on the abscissa. B. Mean channel fluorescence of CD34+ HSC on the ordinate and PMO concentration on the abscissa. The uptake of PMOs is not sequence specific: 20-mer PMOs have similar entry kinetics, and percent-positive CD34+ cells are directly proportional to the PMO concentration in the medium. CD34+ cells were isolated from the blood of healthy subjects by pre-enriching the CD34+ by a lineage negative selection followed by

Figure 2. Time-dependent uptake of FITC-PMO into CD34+ human stem cells. After 3 h at 37C at 150 μg/mL 144-F (a FITC-control PMO), >95% of CD34+ cells became FITC-labeled. At 16 h in culture, the degree of FITC-144-F PMO was the same as the 3-h point. CD34+ cells were isolated and FACS sorted from the blood of healthy subjects as described in

FACS sorting of CD34+ CD45+ cells.

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

Figure 1.

Figure 3. The percent of blood-derived CD34+ cell taking up FITC-PMO. A. Percent-positive (black bar) and mean channel fluorescence (gray bar) CD34+ cells from healthy donors. B. Percent-positive (black bar) and mean channel fluorescence (gray bar) CD34+ cells from diabetic subjects. Uptake is not different in healthy and diabetic subjects, but the rates of maximum saturation of FITC-PMO (measure by MEAN CHANNEL FLUORECENCE) are delayed in diabetic CD34+ cells relative to non-diabetic cells. CD34+ cells were pre-enriched and FACS sorted as described above.

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 retinopathy is expected to be safe and feasible.

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

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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

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,

) inhibitor of TGF-β translation

HSC engraftment while PMO-TGF-β1 does.

TGF-β, but a PMO (5'-GCA CTG CCG AGA GCG CGA ACA-3<sup>0</sup>

c-myc scramble, and c-kit scramble PMO after 3 months post-transplant.
