[CRISPR/Cas9: From research to therapeutic application]. / CRISPR/Cas9 : de la recherche à l'application thérapeutique.

For several decades, genome engineering has raised interest among many researchers and physicians in the study of genetic disorders and their treatments. Compared to its predecessors, zinc-finger nucleases (ZFN) and transcription activator-like effectors (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) is currently the most efficient molecular tool for genome editing. This system, originally identified as a bacterial adaptive immune system, is capable of cutting and modifying any gene of a large number of living organisms. Numerous trials using this technology are being developed to provide effective treatment for several diseases, such as cancer, cardiovascular and ophthalmic disorders. In research, this technology is increasingly used for genetic disease modelling, providing meaningful models of relevant studies as well as a better understanding of underlying pathological mechanisms. Many molecular tools are now available to put this technique into practice in laboratories, and despite the technical and ethical issues raised by manipulation of the genome, CRIPSR/Cas9 offers a new breath of hope for therapeutic research around the world.

The use of radiopharmaceuticals as an effective toxicologic technique for studying nephrotoxicity of drugs: cyclosporine-A.

The concept of altered biologic behavior of administered radiopharmaceuticals is used routinely in clinical nuclear medicine to increase the sensitivity of diagnosis, monitor the efficacy of chemotherapeutic drugs and radiation treatment, and determine injury caused by a drug whose effect has exceeded its therapeutic value. In this study, cyclosporine-A (CsA) an immunosuppressant drug known to cause nephrotoxicity due to tubular impairment and Tc-99m MAG-3, a renal imaging radiopharmaceutical secreted by the tubules have been used in animal models to establish a method for investigating the nephrotoxicity of drugs. New Zealand rabbits and Wistar rats were used. The rabbits and rats were treated with 30 mg/kg of CsA for 4 and 28 consecutive days respectively. Plasma creatinine and urea were measured and renogram studies were performed in the rabbits prior to and on 1, 4, 8, 11 and 15 days after treatment with CsA. For the renogram, the rabbits were given an intravenous bolus injection of 44.4 MBq (1.5 mCi) of Tc-99m MAG-3. The Tmax, T1/2, TTHM and uptake slope of the Tc-99m MAG-3 were calculated. Each rat was injected intravenously with 185 MBq (5 mCi) of Tc-99m MAG-3, killed 3 min later, the kidneys removed and 20 mm frozen sections made. Autoradiograms were generated from the frozen sections. Creatinine and urea levels were also measured in the rats. There was no consistent difference in creatinine and urea levels between control and CsA treated rabbits and rats. However, for the rabbit, on day 1 or 4 after treatment, there was significant increase in the values of Tmax, T1/2, TTHM and uptake slope between the control and CsA treated animals, indicating intrarenal vasoconstriction and delayed transit of Tc-99m MAG-3 from the parenchyma to the collecting system. This delay is dramatically shown in the tissue autoradiograms of the rats. The results are consistent with reported nephrotoxicity of CsA using other techniques. The results of this study, therefore, indicate that the concept of altered biologic behavior of Tc-99m MAG-3 can be used effectively as a toxicologic method for studying nephrotoxicity of drugs as exemplified by CsA.

Experimental studies on multiorgan toxicity of cyclosporine-A in rats using a radiopharmaceutical and comparison with histopathological findings.

Iodine-125 HIPDM was evaluated as a screening agent for studying multiorgan toxicity due to Cyclosporine-A (CsA) and the results were compared to histopathological findings of the tissues. The rats were injected subcutaneously with 50 mg/kg (body weight) of CsA or with equal volume of the vehicle, Cremophor-EL, for 7 consecutive days. A dose of 10 microCi of I-125 HIPDM was injected intravenously at the end of the treatment period. The results indicated that there was a significant increase in the uptake of I-125 HIPDM in the kidney, liver, heart and blood compared to control rats (P < 0.05). However, there was a significant decrease in the uptake of I-125 HIPDM in the spleen compared to control animals (P < 0.001). The lung and brain of CsA treated rats showed no change in the uptake of I-125 HIPDM when compared to control rats. The change in the uptake of I-125 HIPDM in these organs was assumed to indicate tissue response to the toxic effects of CsA. The radiopharmaceutical results were comparable with the histopathological findings in which the organs showed varying degrees of tissue degeneration. It is concluded that the lipophilic radiopharmaceutical, I-125 HIPDM, can be used as an effective screening agent to study multiorgan toxicity due to CsA.

Is cyclosporine toxic to the heart?

Cardiotoxicity caused by cyclosporine was studied in experimental rats by the uptake of radiopharmaceuticals (technetium 99m-labeled pyrophosphate and indium 111-labeled antimyosin) and histologic examination of heart tissues. A dose of 50 mg/kg (body weight) of cyclosporine and an equal volume of vehicle (cremophor-EL) were injected into the rats subcutaneously for 7 or 11 consecutive days. A statistically significant difference (p < 0.05) was noted between the uptake of the radiopharmaceuticals in the hearts of cyclosporine-treated rats compared to the control rats. For 99mTc pyrophosphate, the cardiac uptake ratios of cyclosporine-treated rats to control rats were 2.13 and 4.08 for 7-day and 11-day treatment periods, respectively. For 111In antimyosin, the ratios were greater than 2 for both 7-day and 11-day treatment periods. Histologically, vacuoles were found in single or focal groups of myocytes with interstitial edema in the hearts of cyclosporine-treated rats compared to the control rats. The results of both the uptake of the radiopharmaceuticals and the histologic evidence indicate cell injury in the hearts of cyclosporine-treated rats. Cyclosporine therefore seems to be toxic to the heart tissue.

Effect of cyclosporin A on bile flow in experimental animals using technetium-99m EHIDA.

The effect of cyclosporin A (CyA) on bile flow was studied in experimental rabbits by radionuclide imaging and measurement of bilirubin levels. The rabbits were dosed intravenously with 15 mg/kg CyA for 4 consecutive days. Each rabbit served as its own control. The rabbits were injected IV with technetium (Tc)-99m EHIDA, and dynamic images were obtained prior to and 1, 4, 8, and 15 days after CyA treatment. There was no difference in half-times of blood clearance in control and CyA-treated rabbits. There were differences in the half-times of liver curve and bilirubin measurements between control and 4-day treated rabbits. By the 15th day after CyA treatment both the radionuclide findings and bilirubin levels became normal. The results suggest that CyA causes intrahepatic cholestasis and demonstrate evidence of reversibility of CyA's toxic effect on bile flow after treatment is discontinued.

The use of 125I-HIPDM for studying tissue response due to toxic effects of cyclosporin-A in rats.

125I-HIPDM was used to study the response of various tissues in cyclosporin-A, CyA, treated and control rats. The rats were given 50 mg/kg of CyA for 7 consecutive days. The liver, kidney and heart showed significant increase while the spleen had a pronounced decrease in the uptake of 125I-HIPDM in CyA treated compared to control rats. This difference in the uptake of 125I-HIPDM between CyA treated and control rats is assumed to be the tissue response to toxic effects of CyA. The results indicate that CyA is toxic to liver, kidney, spleen and probably heart. There was no difference in the uptake of 125I-HIPDM in the lung and brain of CyA treated and control rats. This lack of difference is assumed to indicate that CyA does not adversely affect the lung and brain.

Preparation and evaluation of Tc-99m hydroxyethyl starch as a potential radiopharmaceutical for lymphoscintigraphy: comparison with Tc-99m human serum albumin, Tc-99m dextran, and Tc-99m sulfur microcolloid.

Tc-99m hydroxyethyl starch (Tc-99m HES) prepared with a labeling efficiency greater than 95% was evaluated in rabbits for visualization of lymphatic channels and lymph nodes, and the findings compared with Tc-99m human serum albumin (Tc-99m HSA), Tc-99m dextran (Tc-99m DXT), and Tc-99m sulfur microcolloid (Tc-99m SMC). Tc-99m HES showed good visualization of lymphatic channels and regional nodes and it had the highest clearance rate from the injection site (p less than 0.01). Tc-99m HES showed greater uptake by the nodes than Tc-99m DXT (p less than 0.001) at 90 minutes post-injection. The concentration of Tc-99m HES in the lymphatic channels was higher than that of Tc-99m SMC at 90 minutes post-injection (p less than 0.001). Preliminary clinical studies of Tc-99m HES yielded high quality lymphoscintigrams of the leg, and the pelvic and para-aortic lymph nodes in less than 10 minutes post-injection. In addition, partially and completed obstructed lymphatics could be differentiated from normal lymphatic pathways. In conclusion, Tc-99m HES is a promising radiopharmaceutical for imaging of lymphatic channels and nodes.

Is 99Tcm hexamethyl-propyleneamine oxime uptake in the tissues related to glutathione cellular content?

The relationship between the concentration of tissue glutathione (GSH) content and uptake of 99Tcm HMPAO in Sprague Dawley rats was investigated. The GSH content of rat tissue was depleted with diethyl-maleate (DEM) and the ratio of GSH in control to GSH depleted rat was approximately twice that in the brain, liver, kidney, spleen and lung. The GSH content in all the organs studied except the liver had no statistically significant relationship with the uptake of 99Tcm HMPAO. The apparent increase of radioactivity in the liver was due to longer retention of 99Tcm HMPAO. This longer retention was due to stasis of bile flow as confirmed by subsequent experiments in which cholecystokinin (CCK) was administered to GSH depleted rats and compared to the uptake of GSH depleted rats without injection of CCK.

Investigative study of radiopharmaceuticals useful for imaging skeletal muscle injury in experimental animals.

An experimental animal model for studying skeletal muscle injury is described. Tc-99m PYP is accumulated on the skeletal muscle injury, but its uptake on the adjacent bone obscures its usefulness in delineating the extent of the muscle injury. In-111 antimyosin accumulates and delineates the extent of the skeletal muscle injury and does not accumulate on the adjacent bone. Hence, In-111 antimyosin is a good radiopharmaceutical for studying the severity and prognosis of skeletal muscle injury.

Technetium-99m haemaccel: a new lymphoscintigraphic agent.

Haemaccel, a denatured gelatin polymer with average molecular weight of 35,000, was labeled with Tc-99m and evaluated in rats and rabbits. The labeling efficiency was consistently higher than 85%. Tc-99m haemaccel (200 microCi in 0.05 ml) was injected intradermally into the hind footpads of rats lightly anaesthetized with diethyl ether vapour. Using a gamma camera, static images of the anterior view were obtained at various time intervals after injection. The images obtained demonstrated good localization of Tc-99m haemaccel in regional lymph nodes. In rats, about 3% of the injected dose was trapped in the node, and 48% remained at the injection site at 4 h after injection. In rabbits, good-quality images of lymph nodes and lymphatic channels of the hind legs were obtained within 15 min after intradermal injection of Tc-99m haemaccel. Tc-99m haemaccel showed fast migration from the injection site, good accumulation in the lymph nodes, and good visualization of lymphatic channels. Hence, it has a potential application in lymphoscintigraphy.

Comparison of four technetium-99m radiopharmaceuticals for detection and localization of gastrointestinal bleeding in a sheep model.

Four Tc-99 radiopharmaceuticals, Tc-99m sulphur colloid, Tc-99m red blood cells (RBCs), Tc-99m mercaptoacetyltriglycine (MAG3), and Tc-99m DTPA, were studied in an experimental animal model for detection and localization of gastrointestinal (GI) bleeding site in both the upper and lower abdomen. With Tc-99m sulphur colloid and Tc-99m RBCs, it was possible to detect and localize the GI bleeding site in the lower abdomen. With Tc-99m MAG3, it was possible to visualize the bleeding site in both the upper and lower abdomen. However, Tc-99m MAG3 is partially excreted by the liver into the bile, hence it will be difficult to use Tc-99m MAG3 to localize the GI bleeding site in the lower abdomen. With Tc-99m DTPA, it was possible to detect and localize the GI bleeding site simultaneously in both upper and lower abdomen. The overall background radioactivity was reduced considerably by diuresis with frusemide and catheterization of the urinary bladder.

99Tcm hydroxyethyl starch: a potential radiopharmaceutical for lymphoscintigraphy.

The usefulness of 99Tcm hydroxyethyl starch (99Tcm HES) has been evaluated for visualization of lymphatic channels and lymph nodes. 99Tcm HES was prepared with a labelling efficiency higher than 95%. Its usefulness for imaging lymphatic channels and lymph nodes were compared to 99Tcm human serum albumin (99Tcm HSA), 99Tcm dextran (99Tcm DXT) and 99Tcm sulphur micro colloid (99Tcm SMC) in rabbits. 99Tcm HES showed a good visualization of lymphatic channels and lymph nodes and it had the highest clearance rate from the injection site (P less than 0.01). 99Tcm HES showed higher uptake by the nodes than 99Tcm DXT (P less than 0.001) at 90 min post injection. The concentration of 99Tcm HES in the lymphatic channels was higher than that of 99Tcm SMC at 90 min post injection (P less than 0.001). Preliminary studies of 99Tcm HES in humans provided high-quality lymphoscintigrams of the leg, and the pelvic and para aortic lymph nodes in less than 10 min post injection. In addition, partially and completely obstructed lymphatics could be differentiated from the normal one. Hence 99Tcm HES is a potential radiopharmaceutical for visualization of lymphatic channels and nodes.

Development of an animal model using a closed system to study the sensitivity of a radiopharmaceutical for the detection of gastrointestinal bleeding.

An experimental animal model using a closed system to study the sensitivity of radiopharmaceuticals for the detection and localization of gastrointestinal (GI) bleeding site in a sheep was developed. This model was validated with 99mTc-DTPA. Radioactivity as low as 85.47 +/- 13.32 kBq in a volume of 2.1 +/- 0.14 ml at a bleeding rate of 0.07 ml/min was detected. Simulated intermittent bleeding experiments indicated that at 1 h after injection of 99mTc-DTPA there was still enough circulating radioactivity to bleed into the gut and that it was possible to perform repeat injection of 99mTc-DTPA as early as 2 h after the first injection.