Antisense molecules for targeted cancer therapy

The efficacy of traditional anti-cancer agents is hampered by toxicity to normal tissues, due to the lack of specificity for malignant cells. Recent advances in our understanding of molecular genetics and tumor biology have led to the identification of signaling pathways and their regulators implicated in tumorigenesis and malignant progression. Consequently, novel biological agents were designed which specifically target key regulators of cell survival and proliferation activated in malignant cells and thus are superior to unspecific cytotoxic agents. Antisense molecules comprising conventional single-stranded antisense oligonucleotides (ASO) and small interfering RNA (siRNA) inhibit gene expression on the transcript level. Thus, they specifically target the genetic basis of cancer and are particularly useful for inhibiting the expression of oncogenes the protein products of which are inaccessible to small molecules or inhibitory antibodies. Despite the somewhat disappointing results of recent antisense oncology trials, the identification of new cancer targets and ongoing progress in ASO and siRNA technology together with improvements in tumor targeted delivery have raised new hopes that this fascinating intervention concept will eventually translate into enhanced clinical efficacy.

[11C]Choline PET/CT for targeted salvage lymph node dissection in patients with biochemical recurrence after primary curative therapy for prostate cancer. Preliminary results of a prospective study

INTRODUCTION: In this prospective study we set out to investigate the diagnostic value of [(11)C]choline-PET/CT in patients with suspected lymph node metastases before salvage lymph node dissection. PATIENTS AND METHODS: 15 consecutive patients with rising PSA underwent [(11)C]choline-PET/CT and consecutive open salvage pelvic/retroperitoneal extended lymph node dissection due to uptake of [(11)C]choline in at least 1 lymph node. Mean age was 62.1 (range 53-73). RESULTS: [(11)C]choline-PET/CT results were compared with the histopathology reports and clinical follow-up (mean 13.7 months, range 6-24). Mean time to progression was 23.6 months (range 4-81). [(11)C]choline uptake was observed in nodes along the external and internal and common iliac arteries and in the paraaortic region. A positive histology was reported in 8/15 patients. Only one patient had a PSA nadir of <0.1 ng/ml after salvage surgery. Another patient had stable disease with a PSA of 0.5 ng/ml. Three patients developed bone metastases during follow-up. CONCLUSIONS: This interim analysis indicates that [(11)C]choline-PET/CT may be a useful technique in detection of lymph node metastases when rising PSA occurs after definite prostate cancer therapy. The presented cohort is limited in size, but there is still strong evidence that the patients benefit from [(11)C]choline-PET/CT and consecutive salvage lymph node dissection is rather small.

An Innovative Phase I Trial Design Allowing for the Identification of Multiple Potential Maximum Tolerated Doses with Combination Therapy of Targeted Agents

Treatment for cancer often involves combination therapies used both in medical practice and clinical trials. Korn and Simon listed three reasons for the utility of combinations: 1) biochemical synergism, 2) differential susceptibility of tumor cells to different agents, and 3) higher achievable dose intensity by exploiting non-overlapping toxicities to the host. Even if the toxicity profile of each agent of a given combination is known, the toxicity profile of the agents used in combination must be established. Thus, caution is required when designing and evaluating trials with combination therapies. Traditional clinical design is based on the consideration of a single drug. However, a trial of drugs in combination requires a dose-selection procedure that is vastly different than that needed for a single-drug trial. When two drugs are combined in a phase I trial, an important trial objective is to determine the maximum tolerated dose (MTD). The MTD is defined as the dose level below the dose at which two of six patients experience drug-related dose-limiting toxicity (DLT). In phase I trials that combine two agents, more than one MTD generally exists, although all are rarely determined. For example, there may be an MTD that includes high doses of drug A with lower doses of drug B, another one for high doses of drug B with lower doses of drug A, and yet another for intermediate doses of both drugs administered together. With classic phase I trial designs, only one MTD is identified. Our new trial design allows identification of more than one MTD efficiently, within the context of a single protocol. The two drugs combined in our phase I trial are temsirolimus and bevacizumab. Bevacizumab is a monoclonal antibody targeting the vascular endothelial growth factor (VEGF) pathway which is fundamental for tumor growth and metastasis. One mechanism of tumor resistance to antiangiogenic therapy is upregulation of hypoxia inducible factor 1α (HIF-1α) which mediates responses to hypoxic conditions. Temsirolimus has resulted in reduced levels of HIF-1α making this an ideal combination therapy. Dr. Donald Berry developed a trial design schema for evaluating low, intermediate and high dose levels of two drugs given in combination as illustrated in a recently published paper in Biometrics entitled “A Parallel Phase I/II Clinical Trial Design for Combination Therapies.” His trial design utilized cytotoxic chemotherapy. We adapted this design schema by incorporating greater numbers of dose levels for each drug. Additional dose levels are being examined because it has been the experience of phase I trials that targeted agents, when given in combination, are often effective at dosing levels lower than the FDA-approved dose of said drugs. A total of thirteen dose levels including representative high, intermediate and low dose levels of temsirolimus with representative high, intermediate, and low dose levels of bevacizumab will be evaluated. We hypothesize that our new trial design will facilitate identification of more than one MTD, if they exist, efficiently and within the context of a single protocol. Doses gleaned from this approach could potentially allow for a more personalized approach in dose selection from among the MTDs obtained that can be based upon a patient’s specific co-morbid conditions or anticipated toxicities.

APPLICATIONS OF EPHB4 RECEPTOR SPECIFIC PEPTIDES IN TARGETED CANCER IMAGING AND THERAPY

EphB4 receptors, a member of the largest family of receptor tyrosine kinases, are found over-expressed in a variety of tumors cells including glioma cells as well as angiogenic blood vessels. Noninvasive imaging of EphB4 could potentially increase early detection rates, monitor response to therapy directed against EphB4, and improve patient outcomes. Targeted delivery of EphB4 receptor specific peptide conjugated hollow gold nanoshells (HAuNS) into tumors has great potential in cancer imaging and photothermal therapy. In this study, we developed an EphB4 specific peptide named TNYL-RAW and labeled with radioisotope 64Cu and Cy5.5 dye. We also conjugate this specific peptide with hollow gold nanoshells (HAuNS) to evaluate targeted photothermal therapy of cancers. In vitro, 64Cu-DOTA-TNYL- RAW specifically bind to CT26 and PC-3M cells but not to A549 cells. In vivo, Small-animal PET/CT clearly showed the significant uptake of 64Cu-DOTA-TNYL-RAW in CT26 and PC-3M tumors but not in A549 tumors. Furthermore, µPET/CT and near-infrared optical imaging clearly showed the uptake of the dual labeled TNYL-RAW peptide in both U251 and U87 tumors in the brains of nude mice. In U251 tumors, Cy5.5-labeled peptide can bind to EphB4-expressing tumor blood vessels and tumors cells. But in U87 models, dual labeled peptide only could bind to tumor associated blood vessels. Also, Irradiation of PC-3M and CT-26 cell treated with TNYL-PEG-HAuNS nanopatilces with near-infrared (NIR) laser resulted in selective destruction of these cells in vitro. EphB4 targeted TNYL-PEG-HAuNS showed more photothermal killing effect on CT26 tumor model than PEG-HAuNS did. In summary, tumors with overexpression of EphB4 receptors can be noninvasively visualized by micro PET/CT with 64Cu labeled or dual labeled TNYL-RAW peptide. Targeted delivery of TNYL-RAW conjugated HAuNS into tumors can greatly improve the treatment effect of photothermal therapy. The information acquired with this study should be advantageous in improving diagnostics and future applications in photothermal ablation therapy in clinical.

Folate Receptor Targeted Alpha-Therapy Using Terbium-149

Terbium-149 is among the most interesting therapeutic nuclides for medical applications. It decays by emission of short-range α-particles (Eα = 3.967 MeV) with a half-life of 4.12 h. The goal of this study was to investigate the anticancer efficacy of a 149Tb-labeled DOTA-folate conjugate (cm09) using folate receptor (FR)-positive cancer cells in vitro and in tumor-bearing mice. 149Tb was produced at the ISOLDE facility at CERN. Radiolabeling of cm09 with purified 149Tb resulted in a specific activity of ~1.2 MBq/nmol. In vitro assays performed with 149Tb-cm09 revealed a reduced KB cell viability in a FR-specific and activity concentration-dependent manner. Tumor-bearing mice were injected with saline only (group A) or with 149Tb-cm09 (group B: 2.2 MBq; group C: 3.0 MBq). A significant tumor growth delay was found in treated animals resulting in an increased average survival time of mice which received 149Tb-cm09 (B: 30.5 d; C: 43 d) compared to untreated controls (A: 21 d). Analysis of blood parameters revealed no signs of acute toxicity to the kidneys or liver in treated mice over the time of investigation. These results demonstrated the potential of folate-based α-radionuclide therapy in tumor-bearing mice.

Synergism of peptide receptor-targeted Auger electron radiation therapy with anti-angiogenic compounds in a mouse model of neuroendocrine tumors.

BACKGROUND Neuroendocrine tumors are well vascularized and express specific cell surface markers, such as somatostatin receptors and the glucagon-like peptide-1 receptor (GLP-1R). Using the Rip1Tag2 transgenic mouse model of pancreatic neuroendocrine tumors (pNET), we have investigated the potential benefit of a combination of anti-angiogenic treatment with targeted internal radiotherapy. METHODS [Lys40(Ahx-DTPA-111In)NH2]-exendin-4, a radiopeptide that selectively binds to GLP-1R expressed on insulinoma and other neuroendocrine tumor cells, was co-administered with oral vatalanib (an inhibitor of vascular endothelial growth factor receptors (VEGFR)) or imatinib (a c-kit/PDGFR inhibitor). The control groups included single-agent kinase inhibitor treatments and [Lys40(Ahx-DTPA-natIn)NH2]-exendin-4 monotherapy. For biodistribution, Rip1Tag2 mice were pre-treated with oral vatalanib or imatinib for 0, 3, 5, or 7 days at a dose of 100 mg/kg. Subsequently, [Lys40(Ahx-DTPA-111In)NH2]-exendin-4 was administered i.v., and the biodistribution was assessed after 4 h. For therapy, the mice were injected with 1.1 MBq [Lys40(Ahx-DTPA-111In)NH2]-exendin-4 and treated with vatalanib or imatinib 100 mg/kg orally for another 7 days. Tumor volume, tumor cell apoptosis and proliferation, and microvessel density were quantified. RESULTS Combination of [Lys40(Ahx-DTPA-111In)NH2]-exendin-4 and vatalanib was significantly more effective than single treatments (p < 0.05) and reduced the tumor volume by 97% in the absence of organ damage. The pre-treatment of mice with vatalanib led to a reduction in the tumor uptake of [Lys40(Ahx-DTPA-111In)NH2]-exendin-4, indicating that concomitant administration of vatalanib and the radiopeptide was the best approach. Imatinib did not show a synergistic effect with [Lys40(Ahx-DTPA-111In)NH2]-exendin-4. CONCLUSION The combination of 1.1 MBq of [Lys40(Ahx-DTPA-111In)NH2]-exendin-4 with 100 mg/kg vatalanib had the same effect on a neuroendocrine tumor as the injection of 28 MBq of the radiopeptide alone but without any apparent side effects, such as radiation damage of the kidneys.

Evaluation of three different families of bombesin receptor radioantagonists for targeted imaging and therapy of gastrin releasing peptide receptor (GRP-R) positive tumors.

Two new classes of radiolabeled GRP receptor antagonists are studied and compared with the well-established statine-based receptor antagonist DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (RM2, 1; DOTA:1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; Sta:(3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid). The bombesin-based pseudopeptide DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leuψ(CHOH-CH2)-(CH2)2-CH3 (RM7, 2), and the methyl ester DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-OCH3 (ARBA05, 3) analogues are labeled with (111)In and evaluated in vitro in PC-3 cell line and in vivo in PC-3 tumor-bearing nude mice. Antagonist potency was assessed by immunofluorescence-based receptor internalization and Ca(2+) mobilization assays. The conjugates showed good binding affinity, the IC50 value of 2 (3.2 ± 1.8 nM) being 2 and 10 times lower than 1 and 3. Compared to (111)In-1, (111)In-2 showed higher uptake in target tissues such as pancreas (1.5 ± 0.5%IA/g and 39.8 ± 9.3%IA/g at 4 h, respectively), whereas the compounds had similar tumor uptake (11.5 ± 2.4%IA/g and 11.8 ± 3.9%IA/g at 4h, respectively). The displacement of the radioligand in vivo was different in different receptor positive organs and depended on the displacing peptide.

Targeted Gene Repression Technologies for Regenerative Medicine, Genomics, and Gene Therapy

Gene regulation is a complex and tightly controlled process that defines cell function in physiological and abnormal states. Programmable gene repression technologies enable loss-of-function studies for dissecting gene regulation mechanisms and represent an exciting avenue for gene therapy. Established and recently developed methods now exist to modulate gene sequence, epigenetic marks, transcriptional activity, and post-transcriptional processes, providing unprecedented genetic control over cell phenotype. Our objective was to apply and develop targeted repression technologies for regenerative medicine, genomics, and gene therapy applications. We used RNA interference to control cell cycle regulation in myogenic differentiation and enhance the proliferative capacity of tissue engineered cartilage constructs. These studies demonstrate how modulation of a single gene can be used to guide cell differentiation for regenerative medicine strategies. RNA-guided gene regulation with the CRISPR/Cas9 system has rapidly expanded the targeted repression repertoire from silencing single protein-coding genes to modulation of genes, promoters, and other distal regulatory elements. In order to facilitate its adaptation for basic research and translational applications, we demonstrated the high degree of specificity for gene targeting, gene silencing, and chromatin modification possible with Cas9 repressors. The specificity and effectiveness of RNA-guided transcriptional repressors for silencing endogenous genes are promising characteristics for mechanistic studies of gene regulation and cell phenotype. Furthermore, our results support the use of Cas9-based repressors as a platform for novel gene therapy strategies. We developed an in vivo AAV-based gene repression system for silencing endogenous genes in a mouse model. Together, these studies demonstrate the utility of gene repression tools for guiding cell phenotype and the potential of the RNA-guided CRISPR/Cas9 platform for applications such as causal studies of gene regulatory mechanisms and gene therapy.

Development of polymeric drug delivery vehicles for targeted cancer therapy

The aim of my Ph. D. thesis is to generalize a method for targeted anti-cancer drug delivery. Hydrophilic polymer-drug conjugates involve complicated synthesis; drug-encapsulated polymeric nanoparticles limit the loading capability of payloads. This thesis introduces the concept of nanoconjugates to overcome difficulties in synthesis and formulation. Drugs with hydroxyl group are able to initiate polyester synthesis in a regio- and chemo- selective way, with the mediation of ligand-tunable Zinc catalyst. Herein, three anti-cancer drugs are presented to demonstrate the high efficiency and selectivity in the method (Chapter 2-4). The obtained particles are stable in salt solution, releasing drugs over weeks in controlled manner. With the conjugation of aptamer, particles are capable to target prostate cancer cells in vitro. These results open the gateway to evaluate the in vivo efficacy of nanoconjugates for target cancer therapy (Chapter 5). Mechanism study of the polymerization leads to the discovery of chemosite selective synthesis of prodrugs with acrylate functional groups. Functional copolymer-drug conjugates will expand the scope of nanoconjugates (Chapter 6). Liposome-aptamer targeting drug delivery vehicle is well studied to achieve reversible cell-specific delivery of non-hydoxyl drugs e.g. cisplatin (Chapter 7). New monomers and polymerization mechanisms are explored for polyester in order to synthesize nanoconjugates with variety on properties (Chapter 8). Initial efforts to apply this type of prodrugs will be focused on the preparation of hydrogels for stem cell research (Chapter 9).

Upper body musculoskeletal disorders among Australian occupational therapy students

Although upper body musculoskeletal disorders (MSDs) represent an increasingly important issue for university students, few if any studies have targeted the occupational therapy faculty. Given this dearth of information, it was considered necessary to investigate a cross-section of Australian occupational therapy students by means of an established questionnaire survey. Completed replies were obtained from 95.7%, 100% and 97.7% (n = 44, 55 and 48) of students in the first, second and fourth years of a large occupational therapy school in northern Queensland, Australia.---------- The 12-month period prevalence of MSDs was as follows: neck (67.4%), shoulder (46.3%) and upper back (39.5%). Three-quarters of all students (75.5%) reported an MSD occurring in at least one of these body regions. Over half (56.5%) reported an MSD over 2 days' duration in the past year. Almost 40% (39.5%) reported an MSD that had affected their daily life, while one-quarter (25.2%) needed some type of treatment.---------- Logistic regression indicated that students aged over 21 years were almost four times more likely to report shoulder-related MSD (OR 3.7, 95%CI: 1.4-10.2). Year of study in the occupational therapy course was another important MSD correlate, with adjusted odds ratios ranging from 3.3 at the upper back (OR 3.3, 95%CI: 1.2-9.6) to 10.9 at the neck (OR 10.9, 95%CI: 3.2-43.8). Computer usage also incurred a certain degree of risk, with students who spent over 5 hours per week on the computer having an increased risk of MSD at the neck (OR 5.0, 95%CI: 1.3-21.5) and shoulder (OR 4.7, 95%CI: 1.4-18.3).---------- Overall, this study suggests that Australian occupational therapy students have a large burden from MSDs in the upper body region, even more so than other student groups and some working populations. Since the distribution of MSD risk is not uniform among them, interventions to help reduce these conditions need to be carefully targeted. Further longitudinal investigations would also be useful in determining the mechanisms and contributory factors for MSDs among this unique student population.

The effectiveness of a short-term group music therapy intervention for parents who have a child with a disability

The relationship between the quality of parent-child interactions and positive child developmental trajectories is well established (Guralnick, 2006; Shonkoff & Meissels, 2000; Zubrick et al., 2008). However, a range of parental, family, and socio-economic factors can pose risks to parents’ capacity to participate in quality interactions with their children. In particular, families with a child with a disability have been found to have higher levels of parenting stress, and are more likely to experience economic disadvantage, as well as social isolation. The importance of early interventions to promote positive parenting and child development for these families is widely recognised (Shonkoff & Meissels, 2000). However, to date, there is a lack of evidence about the effectiveness of early parenting programs for families who have a young child with a disability. This thesis investigates the impact of a music therapy parenting program, Sing & Grow, on 201 parent-child dyads who attended programs specifically targeted to parents who had a young child with a disability. Sing & Grow is an Australian national early parenting intervention funded by the Australian Government Department of Families, Housing, Community Services and Indigenous Affairs and delivered by Playgroup Queensland. It is designed and delivered by Registered Music Therapists for families with children aged from birth to three years. It aims to improve parenting skills and confidence, improve family functioning (positive parent-child interactions), enhance child development, and provide social networking opportunities to socially isolated families. The intervention targets a range of families in circumstances that have the potential to impact negatively on family functioning. This thesis uses data from the National Evaluation Study of Sing & Grow from programs which were targeted at families who had a young child with a disability. Three studies were conducted to address the objectives of this thesis. Study 1 examines the effects of the Sing & Grow intervention on parent reported pre and post parent mental health, parenting confidence, parenting skills, and child development, and other parent reported outcomes including social support, use of intervention resources, satisfaction with the intervention and perceived benefits of and barriers to participation. Significant improvements from pre to post were found for parent mental health and parent reported child communication and social skills, along with evidence that parents were very satisfied with the program and that it brought social benefits to families. Study 2 explored the pre to post effects of the intervention on children’s developmental skills and parent-child interactions using observational ratings made by clinicians. Significant pre to post improvements were found for parenting sensitivity, parental engagement with child and acceptance of child as well as for child responsiveness to parent, interest, and participation in the intervention, and social skills. Study 3 examined the nature of child and family characteristics that predicted better outcomes for families while taking account of the level of participation in the program. An overall outcome index was calculated and served as the dependent variable in a logistic regression analysis. Families who attended six or more sessions and mothers who had not completed high school were more likely to have higher outcome scores at post intervention than those who attended fewer sessions and those with more educated mothers respectively. The findings of this research indicate that the intervention had a positive impact on participants’ mental health, parenting behaviours and child development and that level of attendance was associated with better outcomes. There was also evidence that the program reached its target of high risk families (i.e., families in which mothers had lower educational levels) and that for these families better outcomes were achieved. There were also indications that the program was accessible and highly regarded by families and that it promoted social connections for participants. A theoretical model of how the intervention is currently working for families is proposed to explain the connections between early parenting, child development and maternal wellbeing. However, more research is required to further elucidate the mechanisms by which the intervention creates change for families. This research presents promising evidence that a short term group music therapy program can elicit important therapeutic benefits for families who have a child with a disability.

Mesenchymal stem cells in osteoarthritis therapy : current status of theory, technology, and applications

Osteoarthritis (OA) is a chronic, non-inflammatory type of arthritis, which usually affects the movable and weight bearing joints of the body. It is the most common joint disease in human beings and common in elderly people. Till date, there are no safe and effective diseases modifying OA drugs (DMOADs) to treat the millions of patients suffering from this serious and debilitating disease. However, recent studies provide strong evidence for the use of mesenchymal stem cell (MSC) therapy in curing cartilage related disorders. Due to their natural differentiation properties, MSCs can serve as vehicles for the delivery of effective, targeted treatment to damaged cartilage in OA disease. In vitro, MSCs can readily be tailored with transgenes with anti-catabolic or pro-anabolic effects to create cartilage-friendly therapeutic vehicles. On the other hand, tissue engineering constructs with scaffolds and biomaterials holds promising biological cartilage therapy. Many of these strategies have been validated in a wide range of in vitro and in vivo studies assessing treatment feasibility or efficacy. In this review, we provide an outline of the rationale and status of stem-cell-based treatments for OA cartilage, and we discuss prospects for clinical implementation and the factors crucial for maintaining the drive towards this goal.