Next-generation muscle-directed gene therapy by in silico vector design.

There is an urgent need to develop the next-generation vectors for gene therapy of muscle disorders, given the relatively modest advances in clinical trials. These vectors should express substantially higher levels of the therapeutic transgene, enabling the use of lower and safer vector doses. In the current study, we identify potent muscle-specific transcriptional cis-regulatory modules (CRMs), containing clusters of transcription factor binding sites, using a genome-wide data-mining strategy. These novel muscle-specific CRMs result in a substantial increase in muscle-specific gene transcription (up to 400-fold) when delivered using adeno-associated viral vectors in mice. Significantly higher and sustained human micro-dystrophin and follistatin expression levels are attained than when conventional promoters are used. This results in robust phenotypic correction in dystrophic mice, without triggering apoptosis or evoking an immune response. This multidisciplinary approach has potentially broad implications for augmenting the efficacy and safety of muscle-directed gene therapy.

Biological carrier molecules of radiopharmaceuticals for molecular cancer imaging and targeted cancer therapy.

Many tumors express one or more proteins that are either absent or hardly present in normal tissues, and which can be targeted by radiopharmaceuticals for either visualization of tumor cells or for targeted therapy. Radiopharmaceuticals can consist of a radionuclide and a carrier molecule that interacts with the tumor target and as such guides the attached radionuclide to the right spot. Radiopharmaceuticals hold great promise for the future of oncology by providing early, precise diagnosis and better, personalized treatment. Most advanced developments with marketed products are based on whole antibodies or antibody fragments as carrier molecules. However, a substantial number of (pre)clinical studies indicate that radiopharmaceuticals based on other carrier molecules, such as peptides, nonimmunoglobulin scaffolds, or nucleic acids may be valuable alternatives. In this review, we discuss the biological molecules that can deliver radionuclide payloads to tumor cells in terms of their structure, the selection procedure, their (pre)clinical status, and advantages or obstacles to their use in a radiopharmaceutical design. We also consider the plethora of molecular targets existing on cancer cells that can be targeted by radiopharmaceuticals, as well as how to select a radionuclide for a given diagnostic or therapeutic product.

Human blood outgrowth endothelial cells improve islet survival and function when co-transplanted in a mouse model of diabetes.

AIMS/HYPOTHESIS: As current islet-transplantation protocols suffer from significant graft loss and dysfunction, strategies to sustain the long-term benefits of this therapy are required. Rapid and adequate oxygen and nutrient delivery by blood vessels improves islet engraftment and function. The present report evaluated a potentially beneficial effect of adult human blood outgrowth endothelial cells (BOEC) on islet graft vascularisation and function. METHODS: Human BOEC, 5 × 10(5), were co-transplanted with a rat marginal-islet graft under the kidney capsule of hyperglycaemic NOD severe combined immunodeficiency (SCID) mice, and the effect on metabolic outcome was evaluated. RESULTS: Although vessel density remained unaffected, co-transplantation of islets with BOEC resulted in a significant and specific improvement of glycaemia and increased plasma C-peptide. Moreover, in contrast to control mice, BOEC recipients displayed reduced beta cell death and increases in body weight, beta cell proliferation and graft-vessel and beta cell volume. In vivo cell tracing demonstrated that BOEC remain at the site of transplantation and do not expand. The potential clinical applicability was underscored by the observed metabolic benefit of co-transplanting islets with BOEC derived from a type 1 diabetes patient. CONCLUSIONS/INTERPRETATION: The present data support the use of autologous BOEC in translational studies that aim to improve current islet-transplantation protocols for the treatment of brittle type 1 diabetes.

Using microdialysis to analyse the passage of monovalent nanobodies through the blood-brain barrier.

BACKGROUND AND PURPOSE: Nanobodies are promising antigen-binding moieties for molecular imaging and therapeutic purposes because of their favourable pharmacological and pharmacokinetic properties. However, the capability of monovalent nanobodies to reach targets in the CNS remains to be demonstrated. EXPERIMENTAL APPROACH: We have assessed the blood-brain barrier permeability of Nb_An33, a nanobody against the Trypanosoma brucei brucei variant-specific surface glycoprotein (VSG). This analysis was performed in healthy rats and in rats that were in the encephalitic stage of African trypanosomiasis using intracerebral microdialysis, single photon emission computed tomography (SPECT) or a combination of both methodologies. This enabled the quantification of unlabelled and (99m) Tc-labelled nanobodies using, respectively, a sensitive VSG-based nanobody-detection elisa, radioactivity measurement in collected microdialysates and SPECT image analysis. KEY RESULTS: The combined read-out methodologies showed that Nb_An33 was detected in the brain of healthy rats following i.v. injection, inflammation-induced damage to the blood-brain barrier, as in the late encephalitic stage of trypanosomiasis, significantly increased the efficiency of passage of the nanobody through this barrier. Complementing SPECT analyses with intracerebral microdialysis improved analysis of brain disposition. There is clear value in assessing penetration of the blood-brain barrier by monovalent nanobodies in models of CNS inflammation. Our data also suggest that rapid clearance from blood might hamper efficient targeting of specific nanobodies to the CNS. CONCLUSIONS AND IMPLICATIONS: Nanobodies can enter the brain parenchyma from the systemic circulation, especially in pathological conditions where the blood-brain barrier integrity is compromised.

Quality control of micro-computed tomography systems.

The rapid proliferation of micro-computed tomography (micro-CT) scanners in preclinical small animal studies has created a need for a method on scanner performance evaluation and scan parameter optimisation. The purpose of this study was to investigate the performance of the scanner with a dedicated micro-CT phantom. The phantom was developed with different independent sections that allow for measurement of major scanner characteristics such as uniformity, linearity, contrast response, dosimetry and resolution. The results of a thorough investigation are discussed.

Influence of heart rate reduction on Doppler myocardial imaging parameters in a small animal model.

In small animals studies, sick animals often have a significant reduction in heart rate while under anesthesia. The influence of heart rate reduction on Doppler myocardial imaging (DMI) parameters is not known. The aim of the present study was to assess the effect of heart rate reduction on DMI parameters in a small animal model. Twenty-four rats underwent transthoracic echocardiography at baseline and during the administration of ivabradine IV. In all rats, left ventricular (LV) systolic velocity, strain and strain rate were measured in the anteroseptal and inferolateral wall segments from short axis views. In 12 rats (group A), M-mode analysis was also performed for assessment of global LV function. In the other 12 rats (groups B), contractility was quantified invasively using the end-systolic pressure-volume relation (ESPVR) and the preload recruitable stroke work (PRSW). During ivabradine, administration heart rate decreased by 18% in group A (p < 0.001) and 36% in group B (p < 0.001). There was a slight increase in LVEDD and LVESD, with no change in cardiac output or LV ejection fraction. During ivabradine administration, DMI parameters did not change significantly in any group. No significant correlation between DMI parameters and heart rate (r(2) = 0.05) or ejection time (r(2) = 0.14) could be found. The absence of changes in contractility was confirmed by the absence of change in PRSW and end-systolic elastance (Ees). In conclusion, moderate heart rate reduction did not influence DMI measurements in this specific rat model. Therefore, in the interpretation of DMI data when performing small animal studies, moderate heart rate reduction does not need to be taken into account.

Doppler myocardial imaging in the diagnosis of early systolic left ventricular dysfunction in diabetic rats.

AIM: To find out if Doppler myocardial imaging (DMI) can detect early signs of left ventricular (LV) dysfunction in a rat model of diabetic cardiomyopathy. METHODS: Eight control and 12 Streptozotocin (STZ)-induced diabetic rats underwent transthoracic echocardiography with high-resolution technology at baseline and 2, 4, 8, 12, and 16 weeks after STZ injection. Radial function was analysed using conventional M-mode, and velocity, strain and strain rate imaging. Longitudinal function was analysed using pulsed Doppler imaging of the mitral annulus. RESULTS: In the diabetic rats, a significant increase in LV end diastolic and end systolic diameter was measured when compared with controls (P < 0.001). Fractional shortening and LV ejection fraction remained unchanged in both groups. Using DMI, diabetic rats demonstrated a decrease in radial systolic velocity (rate of change: +0.01 vs. -0.003 week(-1); P < 0.01) and radial systolic strain rate (+0.003 vs. -0.205 week(-1); P = 0.08) of the anteroseptal wall. Histologic examination revealed dilated cardiomyopathy with no signs of fibrosis. CONCLUSION: Although LV ejection fraction remained preserved, velocity and strain rate imaging was able to detect radial systolic dysfunction in diabetic rats. The absence of histological signs of fibrosis suggests that other mechanisms play a role in the development of diabetic cardiomyopathy.

Synthesis, radiosynthesis, and in vitro characterization of [125I]-2- iodo-L-phenylalanine in a R1M rhabdomyosarcoma cell model as a new potential tumor tracer for SPECT.

[125I]-2-iodo-L-phenylalanine, a new radioiodinated phenylalanine analog was evaluated as a potential specific tumor tracer for SPECT. The tracer is obtained with an overall radiochemical yield of at least 98%, a purity of > 99%, and a specific activity of 11 MBq/mmol in one pot Kit conditions using the Cu1+ assisted isotopic exchange. The tracer is evaluated in vitro using R1M rat rabdomyosarcoma cells in HEPES buffer with and without Na+ ions and in MEM buffer. The uptake of [125I]-2-iodo-L-phenylalanine follows a reversible pseudo-first-order reaction which is the same in presence and absence of Na+ ions, but the compound is not incorporated into the cell proteins. The reversible uptake is proven to occur with the same affinity as L-henylalanine by a saturable transport system which is competitively inhibited by BCH, an L transport type selective molecule. Trans-stimulation of the efflux by BCH and typical L transported amino acids shows that the transporter is of the antiport type and fulfils all the properties of the LAT1 heterodimer transport system. [125I]-2-iodo-L-phenylalanine is thus a phenylalanine analog that for the uptake uses for the major part the LAT1 transport system which is known to be over-expressed in tumor cells. This, together with the easy Kit preparation, makes [123I]-2-iodo-L-phenylalanine a promising tumor specific tracer for SPECT.

Evaluation of the response to therapy of head and neck squamous cell carcinoma by using 3-[123I]iodo-L-alpha-methyl tyrosine and single photon emission tomography.

Early detection of residual tumour tissue offers the possibility for rapid administration of adjuvant treatment. Single photon emission tomography (SPET) with 3-[123I]iodo-L-alpha-methyl tyrosine (IMT) offers the ability to detect recurrence. The aim of this study was to carry out a prospective evaluation of sequential IMT SPET before and after primary therapy and to determine the best timing for scanning in order to establish the response to treatment. Sixteen consecutive patients with histologically proven head and neck cancer (HNC), who underwent IMT SPET before therapy, within 1 week of therapy, and 1 and 3 months after completion of primary therapy were included. Images were classified, according to clinical evaluation, as indicating a high likelihood (HL), intermediate likelihood (IL) and low likelihood (LL) that residual tumoural tissue was present. The definitive clinicopathological diagnosis and follow-up was considered as the 'gold standard'. Based on the definitive clinicopathological outcome, 10 of 16 patients were diagnosed with evidence of local tumour and six without. Nine of 10 patients with evidence of local tumour presented with an HL IMT SPET image after 3 months, seven of whom were from within the first week. In this group, 1/10 patients was considered clinically HS the first week and eventually 4/10 patients became HL, of which there were three at 3 months. Of the six patients diagnosed without local evidence of tumour, with an average follow-up of 15 months, 6/6 were clinically LL in the first week. Three of six had a consistently LL IMT SPET from within the first week. The three other patients had an HL scan the first week, of which one became IL. It is concluded that IMT SPET assessed the response to primary therapy most accurately 3 months after completion of therapy. An IMT SPET image that indicates a high likelihood of residual tumoural tissue may allow earlier stratification of the patients for secondary treatment. If negative, an IMT SPET can exclude residual tumoural tissue from within the first week after completion of therapy.

Diagnosis of recurrent head and neck squamous cell carcinoma with 3-[123I]iodo-L-alpha-methyltyrosine SPET.

The distribution of 3-[123I]iodo-L-alpha-methyltyrosine (123I-3-IMT) in the tumour region of 21 patients with clinically suspected recurrent squamous cell head and neck carcinoma was studied. Single-photon emission tomography (SPET) imaging of the head and neck region was performed 10 min after the injection of 130-170 MBq 123I-3-IMT using a dual-detector gamma camera. Images were interpreted visually and classified as positive or negative for recurrent disease. In addition, target to background ratios (T/B) were measured using semi-automated region of interest analysis. IMT-SPET results were compared with the data derived from clinicopathological follow-up. IMT-SPET detected recurrent disease in 14 of 15 patients (sensitivity 93%). T/B ratios ranged between 1.5 and 2.4 (mean 1.88). One patient with a small tumour (1.2 cm) had a false-negative result. This is attributed to the limited spatial resolution of the SPET system. Five of six patients were correctly diagnosed to be negative for tumour recurrence. T/B ratios ranged between 1.2 and 1.4 (mean 1.30). In one patient IMT-SPET was positive without evidence of recurrence based on clinicopathological follow up. This finding was probably due to uptake into inflammatory tissue. IMT-SPET appears to be a sensitive tool (93%) for the detection of recurrent head and neck squamous cell carcinoma. Further studies with 123I-3-IMT as a metabolic tracer for the detection of head and neck cancer recurrence using SPET are recommended.

In vitro characterization of the influx of 3-[125I]iodo-L-alpha-methyltyrosine and 2-[125I]iodo-L-tyrosine into U266 human myeloma cells: evidence for system T transport.

The aim of this study was to investigate the cellular uptake mechanisms responsible for the accumulation of 3-[(125)I]iodo-L-alpha-methyltyrosine ((125)I-3-IMT) and 2-[(125)I]iodo-L-tyrosine ((125)I-2-IT), two radiotracers for metabolic tumor imaging, using single-photon emission tomography, into U266 human myeloma cancer cells. Time course and concentration dependency of (125)I-3-IMT uptake was assessed. Kinetic parameters were calculated using an Eadie Hofstee plot. A set of competitive inhibitors of the main amino acid transport systems was used for the discrimination of the transporters responsible for the uptake of (125)I-3-IMT and (125)I-2-IT. Protein incorporation of both tracers was determined using acid precipitation. The measured maximum velocity for (125)I-3-IMT transport was 4.199 nmol per mg protein 20 s(-1), and the Michaelis constant was 107.9 microM. Addition of 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH), a competitive inhibitor of System L, reduced the influx by 39.0+/-3.3% for (125)I-3-IMT and 66.3+/-0.9% for (125)I-2-IT. The BCH-insensitive influx was further reduced by Tryptophan (Trp) by 43.8+/-3.5% for (125)I-3-IMT and 15.3+/-1.3% for (125)I-2-IT. This suggests involvement of System T transport. We measured <2% of radioactivity in the acid precipitable fractions of both tracers with no increase in time. We conclude that the influx of (125)I-3-IMT and (125)I-2-IT into U266 human myeloma cells is mediated by both System L and System T amino acid transporters. The kinetic parameters suggest that elevated plasma levels of aromatic amino acids will reduce (123)I-3-IMT uptake in myeloma patients. Both tracers do not enter protein synthesis significantly.