Seven-year follow-up for energy/vitality outcomes in early stage Hodgkin’s disease patients treated with subtotal lymphoid irradiation versus chemotherapy plus radiation: SWOG S9133 and its QOL companion study, S9208

PurposeWe describe 7 years of follow-up for the energy/vitality outcome in early-stage Hodgkin's disease patients treated on a randomized clinical trial that compared subtotal lymphoid irradiation (STLI) with combined modality treatment (CMT) (SWOG 9133). Survivorship research questions involved the extent to which symptoms/side effects endured over a follow-up period of 7 years for this early-stage patient group.MethodsTwo hundred thirty-nine patients participated in the quality of life (QOL) companion study (SWOG 9208) and completed the SF-36 vitality scale, SF-36 health perception item, Cancer Rehabilitation Evaluation System-Short Form (CARES-SF), and symptom distress scale. This paper reports vitality outcome results obtained from randomization, 6 months, and annually for 7 years. To assess changes in vitality over time, we used linear mixed models with patient as a random effect.ResultsPatients receiving CMT had lower observed vitality at 6 months than did the STLI patients (p < .0001). However, beginning at year 1, vitality results did not differ significantly by treatment over the 5-year (p = .13) and 7-year (p = .16) follow-up periods. Vitality only slightly improved over baseline in either group after treatment. The results were similar after accounting for patterns of recurrence and missing data.ConclusionsThis study demonstrated that patients with early-stage Hodgkin's disease experience a short-term (at 6 months) decrease in vitality with treatment, which is more severe with CMT, but that after the first year, vitality scores were similar between the two treatment groups. Enduring fatigue results for patients receiving these therapies were not observed. Implications for cancer survivors These data provide comprehensive 7-year follow-up vitality information, an important symptom for early-stage lymphoma survivors

Tenfibgen Ligand Nanoencapsulation Delivers Bi-Functional Anti-CK2 RNAi Oligomer to Key Sites for Prostate Cancer Targeting Using Human Xenograft Tumors in Mice

<div><p>Protected and specific delivery of nucleic acids to malignant cells remains a highly desirable approach for cancer therapy. Here we present data on the physical and chemical characteristics, mechanism of action, and pilot therapeutic efficacy of a tenfibgen (TBG)-shell nanocapsule technology for tumor-directed delivery of single stranded DNA/RNA chimeric oligomers targeting CK2αα' to xenograft tumors in mice. The sub-50 nm size TBG nanocapsule (s50-TBG) is a slightly negatively charged, uniform particle of 15 - 20 nm size which confers protection to the nucleic acid cargo. The DNA/RNA chimeric oligomer (RNAi-CK2) functions to decrease CK2αα' expression levels via both siRNA and antisense mechanisms. Systemic delivery of s50-TBG-RNAi-CK2 specifically targets malignant cells, including tumor cells in bone, and at low doses reduces size and CK2-related signals in orthotopic primary and metastatic xenograft prostate cancer tumors. In conclusion, the s50-TBG nanoencapsulation technology together with the chimeric oligomer targeting CK2αα' offer significant promise for systemic treatment of prostate malignancy.</p></div

Physiological data for TBG nanocapsules – tissue uptake and analysis of early inflammatory response.

<p>(<b>a</b>) Binding of s50-TBG-RNAi-CK2 to tumor but not liver, spleen and kidney. Tissue sections were subjected to immunofluorescence analysis for Syrian hamster IgG following incubation with s50-TBG-RNAi-CK2. Scale bar 100 µm. (<b>b</b>) Binding of ASOR-DyDOTA to liver but not tumor, spleen and kidney. Tissues were subjected to immunofluorescence analysis for Syrian hamster IgG following incubation with ASOR-DyDOTA. Scale bar 100 µm. (<b>c</b>) Analysis of tibia bone for presence of tumor and uptake of TBG-DyDOTA nanocapsule 24 h following i.p. injection. Upper panels, tumor containing tibia: H&E stain, B = bone, GP = growth plate, M-S = marrow-sinus, T = tumor; immunofluorescence detection of Syrian hamster IgG (green); direct detection of Dy (red); merge of green and red. Center panels, tumor containing tibia: immunofluorescence detection of isotype control for CK8 (green); immunofluorescence detection of CK8 (green); direct detection of Dy (red); merge of green and red. Lower panels, mock injected tibia: immunofluorescence detection of isotype control for CK8 (green); immunofluorescence detection of CK8 (green); direct detection of Dy (red); merge of green and red. DNA counterstain is shown in blue. Scale bars 100 µm. (<b>d</b>) Analysis of liver, spleen and blood for inflammatory response. Immune-competent mice were injected i.v. with 10 mg/kg of s50-TBG-RNAi-CK2 or s50-TBG-sugar or with equal volume vehicle and tissues were collected after 24 h. Liver, spleen and thymus mass are presented relative to the mouse body weight (left axis). Interferon-γ was measured in blood serum (right axis).</p

Nanocapsule-delivered Sleeping Beauty mediates therapeutic Factor VIII expression in liver sinusoidal endothelial cells of hemophilia A mice

Liver sinusoidal endothelial cells are a major endogenous source of Factor VIII (FVIII), lack of which causes the human congenital bleeding disorder hemophilia A. Despite extensive efforts, gene therapy using viral vectors has shown little success in clinical hemophilia trials. Here we achieved cell type–specific gene targeting using hyaluronan- and asialoorosomucoid-coated nanocapsules, generated using dispersion atomization, to direct genes to liver sinusoidal endothelial cells and hepatocytes, respectively. To highlight the therapeutic potential of this approach, we encapsulated Sleeping Beauty transposon expressing the B domain–deleted canine FVIII in cis with Sleeping Beauty transposase in hyaluronan nanocapsules and injected them intravenously into hemophilia A mice. The treated mice exhibited activated partial thromboplastin times that were comparable to those of wild-type mice at 5 and 50 weeks and substantially shorter than those of untreated controls at the same time points. Further, plasma FVIII activity in the treated hemophilia A mice was nearly identical to that in wild-type mice through 50 weeks, while untreated hemophilia A mice exhibited no detectable FVIII activity. Thus, Sleeping Beauty transposon targeted to liver sinusoidal endothelial cells provided long-term expression of FVIII, without apparent antibody formation, and improved the phenotype of hemophilia A mice

Nanocapsule design, morphology, cargo stability, and cargo protection.

<p>(<b>a</b>) Cartoon depiction of nanocapsule design. (<b>b</b>) Transmission electron micrograph of s50-TBG-RNAi-CK2 nanocapsules for <i>in vivo</i> studies. Magnification 230,000×. Scale bar 100 nm. (<b>c</b>) Left panel: Naked RNAi-CK2 oligomer was digested with proteinase K for 24 to 96 h. Inp, undigested input oligomer. Right panel: Naked and s50-TBG encapsulated RNAi-CK2 oligomers were digested with DNase followed by proteinase K as indicated above the panel. Lanes 1 & 2, naked RNAi-CK2; 3 & 4, naked RNAi-CK2 with TBG-sugar nanocapsules included in the digestion; 5 - 7, <i>in vitro</i> use formulation of s50-TBG-RNAi-CK2; 8 – 10, <i>in vivo</i> use formulation of s50-TBG-RNAi-CK2.</p

Characterization of potential mechanisms of CK2 expression downregulation by s50-TBG-RNAi-CK2.

<p>(<b>a</b>) RNase H1 substrate testing of different DNA/RNA composition forms of RNAi-CK2 oligomers. 5′ end-labeled (*) RNA probe was annealed with RNAi-CK2-6R, RNAi-CK2-12R or AS-CK2-PS complementary oligomers, then incubated for various periods of time with RNase H1. The letters on the left-hand side of the gel and inset represent the sequence ladders for the 5′ end-labeled RNA substrate. Sizing ladders are indicated as AL (alkaline lysis), C (minus RNase T1), and T1 (RNase T1 cleavage). RNase H1 digestion times are 0, 30, 60 and 120 s (lanes 1–4, respectively). The identity of the test oligomer is indicated above the lanes. The inset shows a lighter exposure of the RNAi-CK2-12R RNase H1 reaction products. Oligomers complemented with the labeled RNA probe are shown with arrowheads indicating major cleavage sites. For the RNAi-CK2-6R and RNAi-CK2-12R oligomer sequences, lower case denotes 2′ <i>O</i>-methyl RNA residues and capital letters are conventional phosphodiester linked DNA residues. The AS-CK2-PS is a phosphorothioate linked DNA oligomer. (<b>b</b>) Detection of Ago2/RISC cleavage products produced by RNAi-CK2 transfection into PC3-LN4 cells. 5′ RNA ligase-mediated RACE was performed for the CK2α and CK2α' transcripts as outlined in online methods. Lanes 1 & 5, siControl transfected cells; 2 & 6, siCK2 transfected cells; 3 & 7, RNAi-CK2 transfected cells; 4 & 8, no cDNA water controls; M, DNA size markers. The predicted RACE products (CK2α, 242 bp; CK2α', 236 bp) are indicated at left, and the gene specific primers used are indicated above the lanes. (<b>c</b>) Mapping of the RACE cleavage site was obtained by sequencing the products obtained in (b). The mRNA sequence is shown in lowercase, the transfected oligomer is depicted below the mRNA, and cleavage sites are indicated by an arrowhead.</p

Cellular uptake of s50-TBG nanocapsules and effects of s50-TBG-RNAi-CK2 in benign and malignant prostate cells.

<p>(<b>a</b>) Uptake over 24 h of s50-TBG nanocapsules with FeO-dextran cargo in PC3-LN4 cells plated onto TnFn-3D. Cells were stained with DAB-enhanced Prussian blue for iron and counterstained with Fast Red. Scale bar 100 µm. (<b>b</b>) Malignant cell-specific uptake of s50-TBG nanocapsules. s50-TBG-FeO-dextran uptake was determined by iron staining at 8 h in PC3-LN4 and BPH-1 cells grown on TnFn- or laminin-coated nanofiber scaffolds, respectively. Scale bar 100 µm. (<b>c</b>) Cellular proliferation effects of s50-TBG-RNAi-CK2 treatment in benign and malignant prostate cells. PC3-LN4 and C4-2 grown on TnFn-3D and BPH-1 cells grown on laminin-3D in 96-well plates were treated with s50-TBG-RNAi-CK2 or control TBG nanocapsules containing RNAi-RFP-6R targeting Red Fluorescence Protein as indicated. ³H-thymidine was added after 48 h, and cells analyzed at 72 h post-nanocapsule addition. Results are expressed relative to treatment with s50-TBG-sugar nanocapsules. The s50-TBG nanocapsule cargo and cell lines used are indicated below the bars. Means ± SE are presented (n = 3 for all). *p<0.005 relative to s50-TBG-sugar and –RFP; # p<0.01 relative to TBG-sugar; $ p = 0.006 relative to TBG-RFP. (<b>d</b>) s50-TBG-RNAi-CK2 treatment reduced CK2α and CK2α' mRNA steady-state expression levels in PC3-LN4 cells. mRNA isolated from PC3-LN4 cells grown on TnFn 24 and 48 h after s50-TBG-RNAi-CK2 or –sugar treatment as indicated was analyzed by reverse transcriptase real-time quantitative PCR for CK2α and CK2α' expression. HPRT transcript was used to normalize expression levels. Means, SE and p-values are presented (24 h CK2α n = 5, CK2α' n = 6; 48 h CK2α n = 2, CK2α' n = 2).</p

In Vivo Nanocapsule Characteristics and Information.

1<p>Mean ± SD of the average elliptical diameter determined from TEM micrographs (magnification 230,000×) of 20 nanocapsules from two different grid preparations.</p>2<p>Average surface charge measured by DLS from two different preparations across a 20 volt potential in 1 mM KCl at 2 µg/ml. Data shown is the mean ± SE of 15 independent measurements.</p>3<p>Morphology of all nanocapsules determined by visual AFM and TEM observation as uniform, single capsules.</p>4<p>TBG-RNAi-CK2 encapsulation efficiency mean of 79.8±6.1% observed by Burton analysis relative to unencapsulated oligomer.</p>5<p>Upper case letters represent phosphodiester DNA bases; lower case letters represent 2′ <i>O</i>-methyl RNA bases.</p><p>In Vivo Nanocapsule Characteristics and Information.</p