Tracking Gene Expression and Metabolic Processes in Subjects
FIELD OF THE INVENTION
The present invention is of a system, a device and a method for monitoring gene expression and metabolic processes in live subjects, and in particular, of such a system, device and method which uses one of a number of different methods for such monitoring in humans and lower mammals. All of these different methods have the advantage of being non-invasive e.g. without the need to perform a tissue biopsy, yet highly sensitive for the detection of small amounts of biological materials. These methods include the induced expression of the luciferase gene, and the concomitant production of luminescence as the monitored substance. In addition, for monitoring the expression of substantially any native gene in the subject, as well as the expression of inserted genes and/or metabolic processes, PET (positron emission tomography) or MRI (magnetic resonance imaging) can also be used.
BACKGROUND OF THE INVENTION
The development of enhanced biotechnological research methods, the accumulation of disease-related genetic information, and the elucidation of clear clinical objectives, have converged and evolved into the new interdisciplinary field known as gene therapy . Each particular approach to treating a targeted disease must overcome specific obstacles as it moves through the process of concept definition through in vitro and in vivo studies, and pre-clinical safety and efficacy testing and finally to gain acceptance as a treatment for humans "
Researchers working in gene therapy have identified a number of major obstacles related
3 to gene targeting and tissue-specific gene expression , which include: 1 ) developing vehicles for efficient and targeted gene delivery; 2) longevity of gene expression; 3) controlled tissue specific gene expression: 4) simple methods for in vivo assessment of gene delivery and expression; 5) production of high quality and safe clinical grade delivery systems and 6) clear and measurable clinical end points.
Each specific gene therapy project requires a strategic approach built in a stepwise manner. In Figure 1, the concept for strategic planning for gene therapy has been outlined.
This schematic description demonstrates how each particular project can be tailored to meet the general strategy. The first step is to select the most appropriate delivery system from those available or alternatively, to develop one which will better serve as a vehicle to the
targeted organ or cell 4. Step two involves choosing the expression system 5. The expression of a transgene in the targeted tissue/cell depends on specific cellular conditions, which may be influenced by genetic or environmental factors . Thus, the pharmacogenomic approach determines the gene expression pattern in the tissue to be targeted and enables to select for the specific regulatory element that will be used for specific transgene expression. The process of tailoring the specific gene therapy approach depends first of all on verifying which regulatory elements in the targeted gene should be expressed. The third step is essential in determining whether the combination of targeting vehicle and regulatory elements meet the requirements for the specific gene therapy approach which could be detected by CCD, PET or MRI. In the fourth step, viral vectors are produced. This step involves the production of high titer viral batches, without replication competent viruses in a reproducible manner that will meet standard current Good Manufacturing Production (cGMP) requirements.
The development of gene and cell therapy as well as research in the important areas of biological development, cell biology and cancer will be greatly advanced by the capability to determine in vivo the kinetics of organ-specific expression of a transgenes delivered by recombinant viruses, non-viral vectors or by transgene expressing cells 7.
Reporter genes are widely used for monitoring gene expression in prokaryotic, plant and mammalian systems - in cells, reconstituted systems, and whole organisms. The firefly luciferase gene, luc, codes for an enzymatic activity which catalyses the oxidation of luciferin in the presence of ATP to an active bioluminescent substance, thereby generating light. Luciferase is commonly used as a reporter gene due to the availability of highly sensitive detection instruments with fairly low background, which allow a strong signal over background measurement; the gene's relatively short half-life; and its linear response, exceeding six orders of magnitude. In the recent years instrumentation was developed that allow detection of relative minute amount of light emitted from internal organs of mammalian. These technologies are based on the ability of photons to pass through mammalian tissue despite its light absorbing and scattering properties. Another such fluorescent technology involves the imaging of GFP (green fluorescent protein), mainly ex vivo but also in vivo (Yang M, Baranov E. Moossa AR, Penman S. Hoffman RM. Visualizing gene expression by whole-body fluorescence imaging PNAS (USA) 97: 12278-12282. 2000), has been employed for detection of gene expression in living cells and organs.
Recently a new sensitive system was developed for the detection of very low luminescence emitted from cells harboring the luciferase gene (luc) in vivo ' J. The system includes a charged coupled device (CCD) camera in conjugation with a microchannel plate intensifiers which is capable of visualizing low amount of photons emitted by internal mammalian tissue both in vitro and in vivo.
This CCD camera was recently used in a non-invasive manner, to monitor /wc-expressing bacterial pathogens in living mice , as well as real-time expression of luc in transgenic animals. In addition, the camera was used to follow local delivery of the luc gene-expressing element to the lungs by liposomes . Such uses of a CCD camera for detecting luciferase activity have also been described in U.S. Patent No. 5,650,135. However, this camera has not been previously used for the detection of various metabolic processes, and other cellular processes, in living subjects.
In the background art, several detection methods for gene therapy have been developed recently. In addition to the previously described use of GFP and luciferase, as detected with a skin window chamber or other method for detecting light, other methods include MRI 3D imaging with magnetically labeled lymphocytes: imaging of prodrugs (e.g. FIAU labeled with fluorine 18. is F-ganciclovir); and autoquenched near-infrared fluorescence probes 7-9. However all are relatively complex and require further experiments to determine their efficacy.
For example, a method for imaging reporter genes using PET has been previously described, in order to detect the presence of the gene product through the detection of enzymatic activity. Specifically, the presence of the herpes simplex virus 1 thymidine kinase enzyme was detected through the retention of 8-[18F]fluoroganciclovir in liver cells by PET. as a product of enzymatic activity (Gambhir et al., "Imaging adenoviral-directed reporter gene expression in living animals with positron emission tomography, PNAS vol 96, 1999, pages 2333-2338). Unfortunately, this method suffers from the requirement for particular enzymatic activity in order to detect the activity of the reporter gene, such that this activity is only detected indirectly. The major draw back of the HSV-tk technology is that there will be very low incorporation of the [18F]fluoroganciclovir metabolites into the DNA in non dividing cells 17.
However, gene therapy for the correction of defective genes requires both effective gene delivery and assured long-term gene expression. Moreover, in many cases organ specific expression of the transgene is essential. The use of reporter genes is assisted by methods, which have been developed for the delivery of the transgene to the animal including a variety of viral
vectors, non-viral vectors, or in ex-vivo manipulated cells. Unfortunately, no systematic work on the various methods of delivery, organ specificity and duration of expression has been published with regard to the use of such reporter genes. For example, PET has been used to detect the presence of a reporter gene in a construct containing both the reporter gene and a target gene . The reporter gene resulted in the production of an enzyme which altered a PET reporter probe, such that the probe became trapped in the cell and could be detected through PET. However, the reference describes only a very preliminary method, which has not been fully assessed with regard to use with human subjects.
In addition, the system which is described in the above background art reference uses the conventional regulatory elements of the CMV and IRES. Both of these elements have of the drawback of being non specific and inconsistent as related to expression in specific tissues; for example, the CMV promoter has a short half-life in the liver tissue.
Various criteria which are related to the use of reporter genes for real time monitoring of gene therapy and/or other biological processes, such as metabolic processes, in vivo include determination of the efficiency of infection/transfection of various viral and non- viral, delivery methods, promoter specificity and visualization of the ability of the reporter gene and/or its product to reach various organs. In addition, the duration of the expression of the reporter gene as delivered by various vectors utilizing various delivery methods to a variety of organs must also be determined. There is thus a need for, and it would be useful to have, a system, device and method for more direct monitoring of gene activity, metabolic processes and other complex cellular processes in living subjects, particularly humans and lower mammals, through a non-invasive method such as bioluminescence, PET or MRI, for which the efficacy has been determined.
SUMMARY OF THE PRESENT INVENTION
The present invention is of a system, method and device for monitoring gene activity, whether for native or inserted genes, as well as for monitoring metabolic processes and other complex biological processes in subjects, particularly humans and lower mammals. The present invention features a number of different reporting mechanisms for monitoring such biological processes, all of which share the advantage of being non-invasive or at least minimally invasive. Examples of preferred reporting mechanisms include, but are not limited to, the detection of luminescence; detection of positron-emitting compounds with PET (position emission
tomography); and the detection of paramagnetic substances with MRI (magnetic resonance imaging).
As previously described with regard to step three of Figure 1, gene therapy requires a mechanism for continuous in vivo monitoring of targeted gene expression. As described in greater detail below, in the description of the preferred embodiments, the present invention provides an imaging method, system and device which fulfill the requirements for such continuous in vivo monitoring. The method, system and device of the present invention are superior to those which are known in the art, and enable complex metabolic and other cellular processes to be detected, which has not been previously performed in the art. According to the present invention, there is provided a method for monitoring a biological process in a subject, comprising the steps of: (a) administering a substrate or substrates to the subject, the substrate being altered by the biological process; and (b) non-invasively monitoring a change to the substrate to monitor the biological process.
According to another embodiment of the present invention, there is provided a device for monitoring a biological process in a subject after a substrate is administered to the subject, the substrate being altered by the biological process, the device comprising a detector for detecting a change in the substrate caused by the biological process.
Optionally and preferably, the device is part of a system for monitoring a biological process in a subject after a substrate is administered to the subject, the substrate being altered by the biological process.
The term "'native gene" refers to a gene which is present in the subject without external intervention, while the term "inserted gene" refers to a gene which is added to the subject through external intervention.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects, aspects and advantages will be better understood from the following detailed description of a preferred embodiment of the invention with reference to the drawings, wherein:
FIG. 1 is an illustrative algorithm for strategic planning of a specific gene therapy project.
FIG. 2 shows the localization and intensity of light emitted from HepG2//z/c cells in living mice. HepG2 cells were infected with the retroviral vector pLNCLuc expressing the luc gene under the control of a CMV promoter (this work). Transfected cells were subjected to
selection by G418, and stably transfected clones were pooled. 5xl06 HepG2//wc cells were administrated into nude mice either subcutaneously (SC) or intrasplenically (IS). Following the administration of the cells, at the time intervals specified on the top of each picture each mouse tested received an IP injection of 3.3mg luciferin and 0.1ml 4% chloral hydrate. The mice were then placed in a tight light chamber. The photons emitted from the mice were collected and integrated over a period of 20min. Photographing in low intensity light, provided by leaving the chamber door open, generated the gray-scale body image (day 41). The doors were then closed to create a dark environment, permitting the selective detection by the CCD camera system of the photons emitted from the mouse. The intensity of the photon emission was used to create pseudo-color tumor images.
FIG. 3 shows imaging of mice injected with recombinant adenoviruses expressing luc under the control of the HIV-1 LTR constitutive promoter. Recombinant viral particles (1010) carrying the luc under the control of the constitutive HIV-1 LTR promoter were administrated to Balb/C mice either IV or IP. Luciferin was administered and the photons emitted from the mice were detected as described in the legend to Figure 2. Note that in pseudo-color picture taken at 43hr following IV administration of the virus, the two glowing light circles were probably generated by light emitting from the testes.
FIG. 4 shows adenovirus at a titer of 108/ ml (20μl) were applied over the orifice of the mice salivary gland. The luc expression is detected 12 hr after infection. FIG. 5 shows transgenic mice expressing the luc gene under the control of the osteocalcin promoter. The luc activity could be detected at the site of long bone formation and teeth development.
FIG. 6 shows the results of adenovirus administration to the leg muscle of a mice (lOOμl of 109/ml particles). The figure shows light detected at day 6 post-infection. The luc gene is under the control of the SV40 promoter.
FIG. 7 shows luc expression from the leg muscle of a mice following 15 μg pLNCLuc administration to the leg muscle. Expression is still detected at 30 days and over (data not shown).
FIG. 8 shows light emission 45 hr after adenovirus administration (109 particles) into the bladder. In the lower panel demonstrates the emission of light from the bladder after it was excised from the rat pelvis. The two figures at the lower right hand corner of the figure are negative controls.
FIG. 9 shows the results of DNA transfection (100 μg) of a rat bladder harboring a
bladder carcinoma. The luc gene is expressed under the control of the HI 9 promoter. The two small figures at the right hand corner are negative controls.
FIG. 10 shows the results after a human prostate cell line (PC3.38) stably expressing luc from the CMV promoter was implanted orthotopically in the prostate of a SCID mice, thereby inducing the development of metastasis as could be seen in mouse 3 day 44.
FIG. 1 1 shows transgenic mice expressing the luc gene under the control of a liver specific promoter C/EBPP(PLAP) and through a conditional expression system activating the specific liver promoter at the absence of tetracycline (tet) or doxycycline (dox).
FIG. 12 shows the results after T50 mouse bladder cells were stably transfected with the luc gene under the control of a CMV promoter. These cells were subcutaneously implanted in C3H mice. Cells are detected during the growth of the tumor.
FIG. 13 shows imaging of mice injected with recombinant adenoviruses expressing luc under the control of the SV40 constitutive promoter. Recombinant AdGL3Luc viral particles
(1010) carrying the luc under the control of the constitutive SV40 promoter were administrated to Balb/C mice either IV or IP. Luciferin was administered and the photons emitted from the mice were detected as described in the legend to Figure 2.
FIG. 14 shows a schematic diagram of an exemplary flow of operations for PET imaging according to the present invention.
FIG. 15 shows the equipment required for the operations of Figure 14. FIG. 16 shows a graph of the results following administration of a large volume of naked
DNA of pLNCLuc through the tail vein. Such administration directs the DNA to the liver. Without wishing to be limited by a single hypothesis, such direction probably occurs by fenestrating the liver sinusoidal cells covering the hepatocytes, ending in a semi-transfection state. This picture was taken 24h after administration. A similar approach is conducted by injecting the pLNCLuc directly to the brain through blood vessels (veins or arteries) following BBBD (blood brain barrier disruption) with high percent mannitol solution. This method facilitates the high level of expression in the brain from a naked DNA injection.
DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of a system, method and device for monitoring gene activity, whether for native or inserted genes, as well as for monitoring metabolic processes and other complex biological processes in subjects, particularly humans and lower mammals. The present invention features a number of different reporting mechanisms for monitoring such biological
processes, all of which share the advantage of being non-invasive or at least minimally invasive. Examples of preferred reporting mechanisms include, but are not limited to, the detection of luminescence; detection of positron-emitting compounds with PET (position emission tomography); and the detection of paramagnetic substances with MRI (magnetic resonance imaging). Each of these reporting mechanisms is described in greater detail below.
The present invention includes, in a preferred embodiment, the detection of the expression of luminescence in vivo as one tool for monitoring gene activity, metabolic processes and other complex processes in subjects, particularly humans and lower mammals. The luminescence is preferably produced through the activity of the luc gene product, luciferase. which as previously noted, produces luminescence through the consumption of ATP. Alternatively, the promoter specific gene expression imaging method could optionally be performed through the detection of other reporter genes in human subjects such as the green fluorescence protein (GFP). In this case the GFP is constructed downstream from the tissue or pathogen specific regulatory element to be detected. In addition, the present invention features the detection of labeled RNA and/or proteins in a subject, for the detection of native or inserted gene expression. The present invention overcomes problems in the background art, in which imaging of gene expression from whole bodies of animals requires the transplantation of reporter genes and the use of fixed tissue obtained from biopsies. According to preferred embodiments of the present invention for use with bioluminescence. a system including a charged coupled device (CCD) camera in conjugation with a microchannel plate intensifiers, or a cooling CCD system or any other method capable of visualizing low amount of photons (as low as one photon) emitted by internal mammalian tissue i n ι " both in vitro and in vivo, as described in the background art . is suitable for use with the luciferase-based implementation of the present invention. Thus, this bioluminescent detection system is suitable for quantitative monitoring, in vivo (in living mice), organ targeting and real-time kinetics of viral vectors and of ex-vivo manipulated cells in the same mouse continuously over a long period of time. With regard to the present invention, the CCD camera with the microchannel plate intensifiers or liquid-nitrogen cooled CCD has been used to detect luc expression in live mice administered with viral and cellular vectors.
According to another preferred embodiment of the present invention, PET can optionally be used to detect labeled components in RNA and/or protein molecules. As a continuation of the bioluminescent embodiment of the present invention for detecting promoter dependent gene
expression through in vivo continuous luc imaging, the detection with PET has the advantage of detecting specific gene expression, which is promoter dependent. In addition, detection with PET enables gene expression to be detected directly, for example through the detection of the formation of RNA itself. PET utilizes trace quantities of molecules, labeled with short lived positron-emitting radioisotopes. in order to observe and measure rates of biochemical processes in tissues of living subjects, particularly through promoter-dependent activities. Promoter sequences and the factors associated with these promoter sequences are the "engine" recruited for the specific expression of the downstream gene. When such a condition is created following an internal or external signal, a specific RNA template of the gene is produced. The new RNA molecules are made by an enzymatic reaction using nucleosides as building blocks. The nucleosides are small molecules, which enter the cellular environment from the extracellular space. This RNA production process traps nucleoside analogs in the cell, which can then be detected through PET. if these analogs feature at least one positron-emitting atom. In addition, other methods for using PET include the detection of labeled substrates for cellular enzymes or labeled ligands for cellular receptors as PET biomarkers. The intracellular trapping of either biomarkers following an enzymatic reaction or binding to the receptor is detected, quantified and imaged in the living subject through tomography.
The background art had described a method for imaging reporter genes using PET, in order to detect the presence of the gene product through the detection of enzymatic activity. Specifically, the presence of the herpes simplex virus 1 thymidine kinase enzyme was detected through the retention of 8-[18F]fluoroganciclovir in liver cells by PET, as a product of enzymatic activity (Gambhir et al., "Imaging adenoviral-directed reporter gene expression in living animals with positron emission tomography, PNAS vol 96, 1999, pages 2333-2338). However, the background art neither taught nor suggested the use of PET to directly detect gene expression, whether as RNA or protein, as for the present invention. The present invention advantageously uses the expression of RNA molecules and proteins, which incorporate the radioactive substrates, nucleic acids and/or amino acids to form the expressed RNA and produced proteins, thereby resulting in the amplification of the incorporation of the radiolabeled material and of the final signal.
The specific assessment of PET gene expression also optionally and more preferably uses radiolabeled short oligonucleotides to target the specific gene expressed from the "native" or inserted expression system.
MISSING AT THE TIME OF PUBLICATION
Organ distribution of luciferin
For the luciferase light emission reaction, the substrate luciferin has to reach the targeted tissue(s). as the other required substances (oxygen and ATP) are present in each cell and organ. Since luciferin is administrated systemically (126mg/Kg body weight +/- 99%), a critical issue for reliable detection and monitoring of luciferase activity, in whole body scanning, is the determination of whether the luciferin reaches all the organs in the body in a sufficient concentration for the light-generating reaction. The results presented in Table 1 is a summary of the current results, as shown according to the number of the Figure containing these results, and of previously published results. Clearly, luciferin availability, following IP (intraperitoneal) administration of luciferin. and photon detection are not a limiting factor in the use of luciferase activity detection in vivo.
Table 1 : Luciferase/Luciferin activity in organs of living mice
Vectors and methods of delivery
Three vector systems for the delivery of genes into animals were tested: ex-vivo transfected/
infected cells, plasmid vectors and viral vectors. The various vectors were delivered by different methods into different organs.
a) implantation of Luc expressing cells. To follow in vivo cell growth, two animal tumor models were used. Tumorgenic HepG2 cells manipulated ex-vivo to carry the luc reported gene (HepG2//wc) were injected subcutaneously (SC) to evaluate local growth or intrasplenically (IS) to follow metastasis to the liver. The HepG2 cells were prepared by infecting them with a retroviral vector, pLNCLuc, into which the luc gene was inserted under the transcriptional control of the CMV promoter. The drug G418 was used for selection of stably transfected HepG2//-/c cells. An Anthos Lucy Photo-luminometer was used to determine the level of luc activity in cell extracts of individual clones and in extracts of pooled clones (data not shown).
The two tumor models described above were used to monitor the growth and spread of luciferase expressing cells in vivo. To generate the two tumor models, 1.5xl07 /z.c-expressing HepG2 cells, from the pooled retrovirus infected and G418 resistant clones, were administered into nude mice either SC. for local tumor growth, or IS. to generate liver metastasis. At regular intervals (2-3 days), the mice received intraperitoneal (IP) doses of the luciferase substrate luciferin (3.3 mg/mouse, see legend to Figure 2), at which point the CCD camera system was used to measure the resulting bioluminescence. Following SC (subcutaneous) implantation of the HepG2//wc cells, light emitted (Figure 2) was detected from day 9 and at least until day 41 when the experiment was terminated. The increase in light emission between day 9 to day 41 (see pseudo-colors in Figure 2) probably indicates that the transplanted HepG2//z.c cells propagated at their implantation site (Figure 2). After 22 days, the tumors resulting from both the injection of HepG2 cells harboring the luc gene and from the injection of control cells without the luc gene could be palpably detected; however, no light was emitted from the tumor containing the control cells. Thus, the luciferase visualization system enabled the tumor to be detected at least 13 days earlier.
When HepG2//ι.c cells were injected IS (intrasplenically). luc expression in the liver was detected starting at day 22 following injection. This light emission increased over time until at least day 41 when the experiment was terminated. The superimposed presentation (Figure 2, lower right) illustrates that the injected cells did in fact implant and propagate in the liver.
Using the same method of retrovirus infection, stable luc expressing clones of prostate cancer cell line, PC3, and C3H mouse syngeneic T50 bladder cancer cells were prepared (cell
linsPC3.38 and T501uc. respectively).
Similar to the protocol described above, 2X106 of the transfected human prostate PC3.38 cells were orthotopically delivered directly to the prostate of 6-8 weeks old SCID Beige (CB17 Beige 17) mice. The results demonstrate that 19 days after implantation the cells can be seen emitting light from the prostate. The implanted cells grew and emitted light during 54 days after implantation (not shown), when the experiment was terminated. Furthermore, after 44 days metastasis can be noticed spreading to other organs (see Figure 10).
2X10~ T501uc cells were injected subcutaneous to C3H (H2K) female 5-7 weeks old mice. No light could have been detected until day 4 following the implantation of the cells. From day 4 until day 28, at which stage the mice were sacrificed, growing amounts of light was measured in correlation with the growth of the tumor (see Figure 12). To ensure that the light emits from the cancer cells, the tumor was removed on day 28 from the mouse. The tumor emits light outside of the mouse (Figure 12). No light was emitting from the mouse after the removal of the tumor, indicating a full removal of the implanted tumor, at least within the limitation of the number of cells that the camera can detect (data not shown). The low limit of detection of light emitting from cells in a petri dish was determined to be about 500 cells.
These results confirm that this in vivo imaging technology based on the use of the luc reporter gene and the CCD camera can be used to monitor the targeting and gene expression activity of transplanted cells within the same animal over prolonged periods of time.
b) viral vectors
For this experiment, the adenovirus vector harboring the luc gene controlled by the SV40 early promoter was used to define methods of administration of transgenes to the targeted organ as well as localization of the viruses following systemic administration of the viral particles. In addition, the duration of luc expression in the different organs was determined.
As a first step, the minimal titer of injected viruses that results in detectable luciferase activity in-vivo was determined. The titer of Luc expressing virus was determined by end point dilutions of the virus stocks. As little as 10 Luc expressing recombinant adenoviruses were enough to produce light in the liver of the IV infected mouse. Based on these experiments. 108 viral particles were determined as the optimal dose for injection into the mouse for localization of luciferase activity in-vivo.
Luc expression in the liver:
The plasmid AdGL3Luc (constructed for this study) harboring the luc gene under the control of the SV40 constitutive promoter-enhancer from the pGL31uc control (Promega) was transfected HEK293T cells and defective recombinant viruses were collected from the transfected cells and plaque purified. The viruses (1010 viral particles) were injected to Balb/C mice either IP or through the tail vein (IV) for whole body assessment of viral distribution. Following the IV or IP injection of the AdGL3Luc virus, SV40 dependent luc activity could be detected at 17 hours in the liver. The intensity of light emission increased until day 14, at which point it started to diminish. However, the light emission lasted for at least 19 days, at which point the experiment was terminated (Figure 13). This experiment suggests that this in vivo detection system could be used to follow transgene expression in the liver— Furthermore, coupling the expression of the chosen genes to the luc gene either by co-expression on polycistronic mRNAs using an IRES element or by generating Luc fusion proteins will facilitate on-going monitoring of transcription of the transgene in-vivo. Such information could be used in DNA or viral vaccination programs to expedite the generation of individualized vaccination protocols.
Luc expression in the salivary glands:
In another experiment 10 viral particles were introduced by cannulation of the salivary glands via their ducts which open to the mouth. Balb/C (7-8 weeks old) were anesthetized and cannulation of the mouth submandibular gland was conducted under stereoscope using a polyethylene tube to one salivary gland. 12 hours following the direct administration of the recombinant AdGL3Luc virus, luciferin was injected IP (see above) and light was monitored. It is clear from the results presented in Figure 4 that the recombinant virus infected the salivary gland and Luc activity in the gland to which the virus was administrated can be detected..
Luc expression in the bladder of Rats:
Utilizing a catheter, polyethylene PE10. 108 AdGL3Luc virus particles were introduced to the bladder of a male Wistar rat. Light was detected 48h latter. Upon removal of the bladder. only few light emitting cells could have been detected in the whole animal while most of the light emitting cells could have been seen in the removed bladder. Similar results were obtained utilizing a non-viral DNA vector that was introduced to the bladder by the same method (see below).
Luc expression in muscle
Light emission from the muscle was detected 6 days following injection of 108
AdGL3Luc virus particles to the muscle (Figure 6). The lag of 6 days in detectable luciferase activity is quite surprising since introduction of the DNA of this construct by electroporation resulted in luciferase activity in the muscle 24h following the performance of the electroporation procedure (see below).
c) Non viral vectors In several experiments in which 50-100μg naked DNA was injected directly to the tissues, light emission could not be detected. However injection of as little as 3μg of pLNCLuc plasmid DNA into the muscle followed by electroporation (16 pulses at 100V 20msec 500V/99msec, using a Caliper electrodes, BTX electroporator) resulted in light emission from the muscle. Light could have been detected form 24h following electroporation. The light increased with time and was still detectable after 30 days (Figure 7). The expression of luc had been assessed from a tumor specific promoter the HI 9 promoter, which is expressed during fetal development and in various tumor tissues including bladder carcinoma (data not shown). For this experiment, HI 9 promoter elements were adapted and inserted upstream of the luc gene. Introduction, by a catheter, of CaP04/DNA precipitate of plasmid vector pH191uc, in which the luc gene is transcribed from the bladder specific HI 9 promoter, resulted in light emission from the bladder after 30-40h (Figure 9). At 56h following transfection no light was detected. No light was detected in the control experiment in which the plasmid DNA was introduced with no CaPO4 precipitate.
d) Quantification of Luc activity following IV-infection with AdGLSLuc
A whole body screen for light emission was performed up to day 10 following injection of 107 AdGL3Luc defective viruses. Various organs were removed and luciferase activity was determined in tissue extracts (prepared by homogenization by downs in lysis buffer (Promega)). The results presented in Table 2 indicate that only the light emitting organs, liver and stomach exhibit Luc activity in the tissue extracts measured by Lucyl Photoluminometer. (see Table 2). No light was detected in the extracts of spleen, pancreas and intestines.
Table 2. Light detection emitted from whole organs by the photoiuminometer in tissue extracts
The AdGL3Luc defective viruses, at final concentration indicated in the table, were injected as specified, either IV or IP. Organs were removed homogenized and light was determined by Luci 1 photoiuminometer1. No light was detected in the extracts of spleen, pancreas and intestines.
e) Tissue specific expression The AdGL3Luc and the Ad-HIVluc viruses are identical except for the promoters controlling luc gene expression. As shown above (Figure 13), AdGL3Luc, in which luc is under control of the SV40 promoter, was detected in the liver as soon as 17 hr after injection. Under identical conditions, but only about 43 hr following the administration of Ad-HIVluc. in which luc is under control of the HIV-LTR promoter, was detected only in a region over the testis; but no light was detected from the liver (Figure 3). Since the Ad-HIVLuc vector is missing the viral transcription-transactivator gene, the HIV tat the luc activity in the testes must represent the basal promoter activity of the HIV-LTR. As the two adenovirus vectors (AdGL3Luc and Ad-HIVluc) are identical in every respect except the promoters driving the luc gene, the possibility that the AdGL3Luc particles also migrated to the testes but that the faint emission of light from the testis was masked by the strong light emitted from the liver. To test this possibility, the livers were removed from several mice that had been injected with AdGL3Luc, and then the mice were subjected to full body scanning. Even in the absence of the liver, no light emission was detected from the testis (data not shown). These results may suggest that a transcription-activating factor in the testis of mice may activate the HIV-LTR resulting in tissue specific expression of Luc. It should be noted that the HIV-LTR-Luc transcription fusion is
located on an adeno- viral vector and was delivered to the animal via IV injection while imaging of Luc activity driven by the same promoter described previously by Contag et al (1977) was carried out on a luc transgenic mouse. Moreover, in the experimental system, the LTR was not activated.
Organ specific expression in transgenic mice.
Three lines of transgenic mice were used to show that organ specific promoters could drive tissue specific luc expression. The C/EBPβ promoter regulated by the tet-op system was induced by removing dox (doxycycline) from drinking water to express the luc gene in the liver as could be seen in Figure 11. Mice transgenic with the β-lactoglobulin promoter upstream of the luc gene had expressed luc only in the mammary gland. In the transgenic mice with the osteocalcin promoter upstream of the luc gene light was detected specifically in the site of long bone development and teeth as seen in Figure 5.
EXAMPLE 2
Device and Svstem for Monitoring Luciferase Activity The present invention also includes a device and a system for monitoring gene activity, metabolic processes and other complex cellular processes in vivo, particularly for human and lower mammal subjects. This Example describes several preferred but illustrative implementations of such a system and device.
According to a first implementation, which is for direct insertion to the area to be monitored, the system includes a non-directional light probe, which is a globular ending light collector coupled to a light guiding fiber-optic fiber or fibers, whose other end is connected to a single channel light amplifier. The probe end is to be inserted into the organ or tissue or fluid or gas filled structure, the other end is outside and connected to a computer which records the amount of emitted light collected over time. The signal can vary over time. The probe can be inserted percutaneously, intracavitally, through a needle or endoluminally through a guide such as a catheter or endoscope.
According to a second implementation, for internal but indirect detection, the system includes a lens- fiber optic bundle-end for intraluminal emitted light image collection; a third generation micro-channel plate image intensifier (MCP); and a fiber-optic coupler between the image transmitting fiber bundle and the intensifier. The bundle is connected to a CCD camera which is connected to a computer for collecting the image over time. The image can vary over
time. The bundle can be inserted via a needle or trocar or via a catheter or endoscope.
According to a third implementation, for direct surface detection, the system features a lens-MCP -CCD camera to directly collect images of light emitting surfaces or connected to the light output of an endoscope or microscope used in research or for clinical purposes. This implementation is optionally but preferably used for imaging of superficial structures a few millimeters deep: e.g., skin, cornea, retina and other eye structures, nasal, oral, gastrointestinal, peritoneal, genitourinary, biliary, endovascular and endocardial, pial and ependymal surfaces.
The system could also optionally be used for artificially created surfaces as in endoscopic surgical procedures. According to a fourth implementation, for deeper structures or monitoring of volumes of tissues, the system includes a MCP-CCD camera with either a regular optical lens for a regular single projection image. This camera is connected to a computer recording the emitted image over time. The system is optionally used for deeper light emitting structures, such as heart, liver, brain, and other organs for example. This system can also optionally be implemented with an array of such cameras surrounding the patient or object to be imaged in the number of projections desired. This array is connected to a computer recording all the projection images over time.
According to yet another embodiment of this system, it can be implemented with a fiber-optic array forming a cylindrical light collecting surface surrounding the emitting patient or object to be imaged. This whole array is then connected at the other end to an MCP-CCD camera, which is connected to a computer giving a flat time-varying cartographic map of all the projections. This is also the most time efficient way to collect the emitted light.
According to a fifth implementation of the present invention, the system is optionally implemented with tomographic imaging. Briefly, the "surround" image obtained from the fiber-optic array, as described above, is used to reconstruct on the computer a time varying 3D image using algorithms similar to SPECT. This implementation is optionally used for the imaging of deeper light emitting structures or structures with complex morphology. The system may optionally feature a rotating camera, or static detector array ring.
According to a sixth implementation of the present invention, light can optionally and specifically be detected with particular surgical and/or diagnostic instrumentation. Examples of such instrumentation include, but are not limited to:
1. Laparoscopy for detection of light from internal cavities in the body. This could include the pleural, peritoneal and other closed body spaces.
2. Arthroscopy for detection of light from articular spaces, such as the knee.
3. Bronchoscopy for detection of gene expression by light from the internal parts of the lung.
3. Endoscopy and Colonoscopy for detection of light for gene expression from the gastrointestinal tract.
4. Funduscopy for detection of light in the eye.
5. Angioscopy for detection of light inside the arteries, veins and heart.
6. Cystoscopy for detection of light inside the bladder.
The sixth implementation of the present invention is particularly useful for surgical applications, although of course other implementations of the present invention could also be used for such applications. One of the main problems in surgical oncology is to be able to distinguish between tumor and non tumor in the operating room in "real time" while performing surgery on the patient. Until now, surgeons have been using repeated histological analysis of frozen section while operating in an attempt to overcome this problem. However, this method is time consuming, since the surgeon must wait for the pathological analysis following each biopsy which is taken. Surgeons have also used intraoperative ultrasound methods.
By contrast, the method of the present invention, through specific tumor expression sequences, enables tumor and non tumor tissue to be distinguished in situ in the operating room itself in "real time", through the use of the CCD camera for light emission. As long as light is emitted, tumor tissue is still present at the site and needs to be excised. In a similar fashion, the luc gene could optionally be detected at other diseases sites, or in surrounding tissue, such as in the case of abscesses or other types of infections, or other pathological conditions to be treated surgically in the operating room.
For any of these implementations or embodiments, optionally and preferably scatter is decreased through the use of a narrow energy window in camera (detector array) or use of multi-frequency light emission.
Preferably, the imaging is performed in a space, such as a room, which is totally or at least substantially dark in the spectrum used for imaging. For the human operator of the system, visibility can optionally be provided by using lamps with visible light which does not interfere with luciferin frequency, or alternatively with non-visible light and goggles for seeing ultra violet or infrared light. Infrared light can optionally be natural heat emitted by bodies and objects in the room.
These systems are preferably monitored through the detection of luminescence from
luciferase activity. Exemplary uses of the system of the present invention include monitoring general metabolic activity, viability, energetic, state and response to various interventions and environments (hypoxia ischemia, and so forth). This is in addition to monitoring expression and activity of specifically targeted genes. The administration of luciferin is optionally performed through a number of routes, such as IV (intravenous). IA (intra-arterial), PO (orally), topical, and inhaled. Direct injection or infusion may optionally be performed intra luminally; for example, through CSF (cerebrospinal fluid), biliary or genitourinary system, lymphatic system, intraperitoneal. and so forth, as well as direct extracellular space injection or infusion in solid tissues. According to optional but preferred embodiments of the present invention, specific localization of the site or sites within the subject from which light is emitted is performed through a combination of the detection of light and a structural imaging technology. This combination enables the specific tissue at which the gene is expressed to be located. The human body internal structures are optionally and more preferably imaged by computer tomography (CT) or magnetic resonance imaging (MRI). Both of the light and MRI or CT technologies compliment each another for functional and structural information, respectively.
According to still other optional but preferred embodiments of the present invention, a therapeutic gene is preferably conjugated to the luc gene. As indicated previously, the pharmacogenomic approach is used for the present invention to detect light at the site(s) where genes are expressed. This method enables disease-associated genes to be detected. Gene expression supports the diagnosis of such conditions and monitoring of follow-on therapy. In addition, a therapeutic gene can itself be fused to the luc gene, such that the combination would only need to be administered once. The result suggests that after a successful therapeutic effect. light is no longer emitted from the diseased tissue. In cases without any therapeutic effect, or with an effect below a minimum level, the luc gene is active and light is detected.
EXAMPLE 3 A Light Gene Imaging Kit According to previously described embodiments of the present invention, the detection of the activity of the luciferase gene is used for diagnosis of various pathological conditions, for monitoring the progress of various types of therapeutic modalities such as gene therapy for example, and for locating particular types of tissues and/or tissue activities in "real time", as well as for the non-invasive monitoring of metabolic and other biological processes. According
to a preferred but optional embodiment of the present invention, as described in this Example, these various implementations and embodiments are preferably assisted through the provision of a luciferase light imaging kit.
The luciferase light imaging kit (LLIK) for human diseases optionally but preferably includes a number of components. For example, the luciferin substrate is preferably provided in bottles. The amount of luciferin to be administered to human subjects is preferably from about 1 to about 50 mg/kg body weight depending on the type of tissue which needs to be assessed.
The luc gene, causing the expression of any possible type of light (e.g. red, yellow, green), with the requisite upstream tissue/cell specific promoter to be administered, is also preferably provided. The gene expression cassette is optionally and preferably prepared according to any suitable delivery vehicle. Non-limiting examples of such vehicles include administration as naked DNA, the use of liposomes as the vehicle, delivery in other non-viral vectors or in viral vectors, and delivery in cells. The amount of DNA is more preferably between 1 μg/kg to 1000 μg/kg body weight in the case of naked DNA or non viral vectors. In the case of viral vectors, the viral titer is more preferably between 104 to 1013 viral particles per Kg body weight.
Alternatively the luc gene is optionally and preferably fused to a therapeutic or other marker gene. The fused gene is then provided in the kit.
EXAMPLE 4
Use of Positron Emission Tomography in the Detection of Gene Expression through Measurements of RNA and Protein Biosynthesis
This section describes another exemplary implementation of the present invention, using PET (positron emission tomography). PET has some unique features for accurate and noninvasive detection of the in vivo concentration of radio pharmaceuticals tagged with positron emitters. Some of these features, such as stimulation by the emission of two gamma rays at 51 IkeV, following positron annihilation and coincidence detection of the two gamma rays for better spatial localization of the positron decay, and accurate correction for self absorption of gamma rays in the body, make PET ideal for accurate reconstruction of radio-nucleotide distribution in the body using computed tomography principles. PET, using tracers of tumor metabolism, has been utilized in the evaluation of treatment response in a variety of tumors and therapeutic response, indicating that these tracers measure useful parameters for the assessment
of therapeutic efficacy. According to the present invention, the use of PET biomarkers such as labeled nucleic acids and amino acids could enable the PET methodology to be used in order to monitor gene therapy.
More generally, PET could be used to detect the expression of a native or inserted gene in a subject. By detection of expression, it is meant that the presence of RNA and/or protein from the expression of the gene is detected. Furthermore, such detection may optionally be used to determine the presence of the native or inserted gene, and/or to detect the presence of a particular metabolic process in the subject. Preferably, such a presence is detected with PET with a radiolabeled RNA or protein component, such as radiolabeled ribonucleotide or a radiolabeled amino acid, for example. Both implementations are described in the flow diagram of Figure 14.
Briefly, as shown in Figure 14, DNA and or RNA transfection, infection or naked DNA administration is preferably performed, in order to provide a particular promoter for driving the production of RNA or protein. Next, either a radiolabeled RNA component, such as the nucleotide 5-FU; a radiolabeled amino acid (whether naturally occurring or synthetic); or a unique radiolabeled amino acid such as selenocysteine or selenomethionine is injected in combination with the same promoter element to express the specific tRNA. This results in the accumulation of a specific RNA and/or protein, through a promoter-dependent effect. This specific RNA and/or protein is radiolabeled with an appropriate radioactive label, and thus can detected through PET.
Human gene expression imaging by tracking RNA production
In each cell type and in particular in every physiological or pathological condition specific genes are expressed. Gene expression is dependent on the promoter sequences, which respond to the summation of the nuclear environmental activation to suppression balance. Various factors, including transcription factors, cell cycle dependent proteins, short RNA molecules and other unknown agents control gene expression through promoter and enhancer interactions.
Figure 14 shows a systemic method of gene targeting technologies, by viral vectors or other carriers, or even direct administration of DNA or RNA, as shown. Figure 15 shows the equipment which is required for the operations shown in Figure 14. The DNA construct is composed of three components: A. The promoter region that is responsible for the specificity and level of expression. This sequence is tissue specific and or disease specific. B. The reporter
-n
transgene. This is an RNA molecule which is not translated to a protein, or an RNA stretch translated to a stable or a less stable protein. C. RNA stability sequences, which are usually located at the 3 ' of the RNA. In cases that a unique amino acid is administered to the subject, a specific tRNA is also required as part of the transgene. As shown, the expression construct is preferably administered with one or more of a labeled nucleoside, nucleotide or oligonucleotides. The use of an oligonucleotide may optionally and preferably be used in order to increase specificity of labeling. The expression construct is targeted to the nucleus. The nucleoside, nucleotide or oligonucleotide analogs are integrated into the RNA transcribed from the DNA construct. The nucleoside, nucleotide or oligonucleotide are labeled with either carbon- 1 1, iodine-131 (for SPECT), fluorine-18, bromine-76 or any other positron emitting radioisotopes.
Optionally and preferably, labeled 5-fluoro uracil (5-FU*) is administered, as an example of one of the building blocks of RNA. The labeled 5-FU* is integrated into the specific promoter dependent expressed RNA. The labeled RNA* is accumulated in the nucleus and exported to the cytosol. In the cytoplasm the labeled RNA* is also accumulating to high levels dependent on the promoter activity and the 3' stability sequences. Each cell encounters high level of positron emission, to be detected by PET.
Human gene expression imaging of Protein The building blocks of RNA could be detected when labeled with Positron Emitter
Nuclides as described previously. In a similar fashion, amino acids (aa*), which are the building blocks of proteins, can also optionally be so labeled. The specific expression vector with the specific promoter generates the expression of the specific RNA, which is translated to the reported protein. Administration of labeled aa* will integrate into the high level expressed reported protein. This protein, dependent on the protein half-life, time is accumulated in the cytosol. This labeled, accumulated and reported protein is than detected by the PET, as shown in Figure 14. Manipulations of the protein sequence determine the protein half-life time, compartmentalization, and if the protein is accumulated in the cytosol or secreted. In some cases specific tRNA's for rare amino acids such as selenocysteine, if given as at least one of the radiolabeled amino acids, are also administered as RNA or as expression cassettes to enable the integration of the specific amino acid to the protein structure. The incorporation of the radiolabeled amino acid into the protein structure enables the specific site of gene expression to be detected within the subject.
Specific Example of Detection of Gene Expression
The following example provides a non-limiting, illustrative description of the use of PET for detect gene expression. Mice are infected i.v. with a replication defective adenoviral vector harboring the H19 gene or albumin are expressed under a liver specific promoter (o-iAT). At day two following the administration of 1x10s transducing viral particles per mice, 5-18FU is injected to the animals. To an additional group of mice, one day after the administration of 5-18FU. a radiolabeled amino acid (carbon- 1 1 methionine) is also administered. The mice are followed for liver specific HI 9 or albumin gene expression. Gene expression of the RNA, and/or the RNA and protein levels, are detected using the PET technology.
EXAMPLE 5 A PET Gene Imaging Kit According to previously described embodiments of the present invention. PET is used to detect the expression of a native and/or inserted gene in a subject. According to the present invention, the use of PET biomarkers such as labeled nucleic acids and amino acids could enable the PET methodology to be used in order to monitor gene therapy. According to a preferred but optional embodiment of the present invention, as described in this Example, these various implementations and embodiments are preferably assisted through the provision of a PET gene imaging kit.
The PET kit optionally and preferably includes the expression vector as required. The expression vector is optional since in some situations, the expression is dependent on endogenous genes of host or pathogen. The contrast material includes nucleic acids (Adenine ,Thymine, Uracil. Guanine or Cytosine) or one of the amino acids (natural or synthetic), such as selenocysteine for example, which are labeled with positron emitter radio-nucleotide. The kit optionally and more preferably features the following components: 1. Specific promoter dependent expression vectors, with RNA stabilizing sequences derived from ribosomal RNA's or other needed sequences to determine the longevity of expression. The expression vector is preferably DNA or RNA. In addition, in specific cases the expression vector is encapsidated in viral vectors e.g. retroviruses, lentiviruses, SV40, adenovirus, AAV, or is entrapped in specific deliver systems as liposomes or carbohydrates. For the incorporation of specific synthetic and/or rare amino acids, such as selenocysteine for example, an additional tRNA is also delivered.
2. The second component in the PET imaging kit is the radioactive material. This could include one of the nucleic acids as described above or one of the amino acids, natural or synthetic, such as selenocysteine for example. The timing of injection of the expression method and the radioactive material is dependent on the tl/2 (half-life) of both the activity from the expression method and the radioactive material itself. In some cases radioactive nucleic acids as well as the radioactive amino acids are injected simultaneously. In other cases radioactive lipids and carbohydrates are generated to become radioactive material as substrates for post-transcriptional processing of proteins as in the case prenylation (covalent attachment of an isoprenoid). Furthermore, short 2-50 mer radio-oligonucleotides are used to target specific RNA's expressed in specific tissues. These short radio-oligos improve specificity.
Therefore, the second component may optionally and more preferably contain one of the following components ("contrast material"): 1. Radio-nucleic acids; 2. Radio-amino acids, or 3. Radio-oligonucleotides.
3. The kit preferably also includes instructions for use of any of the above combinations or each separately.
EXAMPLE 6 Use of MRI in the Detection of Gene Expression through Measurements of RNA and Protein Biosynthesis
This section describes another exemplary implementation of the present invention, using MRI (magnetic resonance imaging) for molecular imaging. Molecular imaging is the imaging of the anatomic and cellular distribution and concentration of specific molecules. MRI is sensitive to multiple parameters: Tl, T2, T2* (relaxation times of the hydrogen proton magnetization), proton density (concentration of hydrogen protons), H (hydrogen proton) spectrum, diffusion (a measure of the diffusion rate of water molecules), magnetization transfer (a measure of interaction between the hydrogen protons of free water and those of immobile macromolecular structures; e.g., myelin and collagen) and ; macroscopic, microscopic and molecular motion & structure. Bo (strength of the main magnetic field in different regions, which may also be affected by paramagnetic and susceptibility properties of tissues and anatomy) and so forth.
MRI based molecular imaging can utilize intrinsic or extrinsic contrast mechanisms. The most effective contrast mechanism, both intrinsic and extrinsic, is provided through
paramagnetic species that have their unpaired electrons close enough to water molecules to affect the magnetic resonance relaxation times of these water molecules. Native paramagnetic molecules include melanin, deoxyhemoglobin, methemoglobin, ferritin, hemosiderin. manganese. Non-limiting examples of native Η spectrally unique molecules include fat, creatinine, choline, lactate, and so forth. The most common extrinsic paramagnetic entities used for imaging are gadolinium-based agents. Other agents include iron and manganese. Both native molecules and reporter genes can optionally be so imaged according to the present invention.
To be imaged, the targeted native molecule must have unique native MRI contrast or must interact selectively with an extrinsic contrast agent. Fat. hemoglobin and its degradation products, and melanin are intrinsic molecules with unique native MRI contrast. Otherwise, an extrinsic contrast-agent must interact with the molecular moiety to be imaged. This interaction is based on the formation of an interaction with sufficient affinity to be selective.
One optional implementation of such an extrinsic agent can preferably be provided through the attachment of an antibody specific to the target molecule to the extrinsic paramagnetic contrast agent. The affinity can high enough to be considered permanent, or may be loose enough to be in competition with other moieties. In competitive binding between similar moieties, it might be easier to label one such moiety, although the concentration or activity of a second moiety may additionally or alternatively be of interest. In such a situation, there is an inverse relation between the concentration of bound labeled moiety and the concentration of the moiety of interest.
Competitive association may be used to indirectly detect the presence of the competing molecule. If the targeted native molecule has enzymatic properties, the extrinsic contrast agent can optionally be a paramagnetic agent, such as a gadolinium-based agent with addition of the enzyme substrate to hinder paramagnetic interaction with water molecules. Enzymatic action of the target molecule would then remove or sterically deform the substrate that is attached to the contrast agent, exposing its paramagnetic center to interaction with surrounding water molecules. In other words, the target molecule would "turn on" the inactive extrinsic contrast agent. Extracellular target molecules that have local distribution or are attached to cell surfaces are more easily accessed by extrinsic contrast agents. Intracellular target molecules can optionally be accessed by extrinsic contrast agents that can enter the cell, which requires the agent to have properties that allow cellular uptake. These properties may be specific for certain
cell populations; e.g.. hepatocyte. reticuloendthelial cell, glial cell, blood brain barrier endothelial cells, and so forth.
For reporter gene imaging, preferably the reporter gene produces a target molecule that is unique or in high enough concentration to allow a high target to background ratio. The reporter gene product may be intracellular. attached to the cell surface or extracellular. It may have intrinsic paramagnetic properties, for example melanin, or have a unique Η spectrum, or it may be targeted by an extrinsic contrast agent as discussed above for native molecular imaging.
For example, the melanin gene could optionally be inserted as a reporter gene molecule, after which the characteristic intracellular Tl shortening effect on Tl weighted MRI could be observed.
Preferably, antibodies or binding substrates, such as transferin, to the surface antigen or binder used as the reporter gene, such as the transferin receptor, are labeled with gadolinium or superparamagnetic iron particles. The reporter gene is inserted. Next, the labeled agent is injected intravenously and imaged with the appropriate MRI sequence; e.g., Tl weighted images for gadolinium agents and T2* for the superparamagnetic iron particles. The characteristic Tl or T2* shortening in the area of uptake is then detected.
According to preferred embodiments of the present invention, a gadolinium chelate is synthesized with an attachment that hinders interaction with free water protons. This attachment is cleavable by a specific enzyme (carbohydrate or protein type). This enzyme is produced from the reporter gene which has been inserted into the desired location in the subject. The hindered gadolinium chelate is then injected, and Tl weighted MRI is used to look for Tl shortening that indicates the presence of the reporter gene via the specific enzymatic activation of the gadolinium chelate.
EXAMPLE 7
A MRI Imaging Kit According to previously described embodiments of the present invention, MRI is used to detect the presence of a native molecule and or the product, whether RNA or protein, of a reporter gene in a subject. According to preferred embodiments of the present invention, the use of a suitable contrast agent could enable the MRI methodology to be used in order to monitor gene therapy. According to a preferred but optional embodiment of the present invention, as described in this Example, these various implementations and embodiments are preferably assisted through the provision of a MRI imaging kit.
The MRI imaging kit optionally and preferably contains the following components. MRI contrast material such as the Gadolinium -chelator complex (GCC) is preferably conjugated to.one or two components. GCC is optionally and preferably conjugated to a short stretch of hydrophobic peptides enabling the complex to enter cells or a sequence derived from the HIV tat (~1 1 amino acids). In some cases this peptide incorporates also specific sequences for targeting e.g. ACTH 1-39, Angiotensin II fragment 3-8, ANP fragment 3-28, CD 4 fragments, Erythropoietin fragment 1-26, and other receptor specific binding fragments (Watson K, Edwards RJ. HIV- 1 -trans-activating (Tat) protein: both a target and a tool in therapeutic approaches. Biochem Pharmacol; 58:1521-8, 1999). The combination of the two components enables specific binding, and then intemalization of GCC, into cells. The amount of unmodified GCC to be injected is more preferably in the range of from about 0.01 to about 1.0 mmol/kg for a human subject. Since the GCC may be modified with the cell binding and fusion components, the amount of material to be injected is more preferably determined for each specific situation, primarily according to the type of tissue which is expected to detect the contrast material. The GCC-peptide complex, with or without the specific cell membrane binding and intemalization domains, is then optionally and more preferably bound to a GCC complex covering protein. This covering protein is composed of human polypeptide incorporating specific proteolytic sites. In the cell, an in situ and/or a targeted protease encoding gene is optionally and more preferably expressed from specific regulatory sequences activated in the specific disease or physiological condition. For example, since some tumors express the HI 9 gene, the HI 9 minimal promoter would optionally and preferably be used to drive the expression of a known cellular protease for these tumors. If internalized into the cell, the GCC complex undergoes an uncovering process by cellular or pathogen proteases. This uncovering process cleaves the covering protein and therefore uncovers the GCC agent for detection by MRI imaging technology. Thus, the site of gene expression is determined through the MRI imaging technology.
The kit optionally and most preferably also includes the GCC modified gene expression reagent as the contrast material, and disposable equipment for systemic or local injection of the contrast material.
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