Patent Description:
The present disclosure is in the field of reagents for use in evaluation of bioluminesnce in a living organism. In particular, described herein are methods for normalizing the bioluminescent signal observed from an organism containing a bioluminescent protein using a fluorescent dye.

Detection of light from transgenic animals carrying a gene encoding a light-generating protein is a powerful tool in diagnostics, drug discovery and medicine that allows for the identification of disease pathways, determination of mechanisms of action, evaluation of efficacy of drug compounds, and monitoring lead candidates' effects on disease progression in living animals. See, e.g., <CIT>; <CIT>; <CIT>;<CIT>;<CIT>; <CIT>;<CIT>; <CIT>;<CIT>; <CIT>; and <CIT>.

In the case of bioluminescent proteins a substrate is typically administered to the animal prior to the evaluation. For example, luciferase (e.g., encoded by eukaryotic luc gene), catalyses the oxidation of D-luciferin (D-(-)-<NUM>-(<NUM>'-hydroxy-<NUM>'benzothioazolyl)thiazoline-<NUM>-carboxylic acid) in the presence of ATP to generate light signals. The availability of the substrate has been shown to effect photon emission efficiency. See, e.g., <NPL>; <NPL>. Various derivatives of luciferin have been prepared, including preparations in which luciferin is covalently bonded to a targeting moiety (see, e.g., <CIT>) or a fluorescent label (see, e.g., <NUM>-fluoroluciferin available from Promega) as well as <NUM>-substituted D-luciferin esters for use evaluation of pesticides (see, e.g., <CIT>). <CIT> describes the use of the combination of fluorescence and luminescence measurement to identify the stage of the cell cycle in living cells into which a gene to be analysed is introduced as well as the amount of expression of the gene. This document also discloses administering a complex of a pathogen, a fluorescent component, and a luminescent component to an animal.

Despite the wide-spread use of bioluminescent imaging techniques, there remains a need a need for improved methods for detecting, quantifying and validating bioluminescence in living animals.

The present invention includes methods for evaluating and quantifying bioluminescence in a living animal by providing a tracking dye that co-distributes with the bioluminescent substrate administered to the animal.

Described herein is a method of normalizing a bioluminescent signal detected in a live animal comprising a bioluminescent protein by administering a composition comprising a bioluminescent substrate (e.g., luciferin) and a tracking dye (e.g., a fluorescent tracking dye) as described herein to the animal, measuring the bioluminescent signal generated by reaction of the bioluminescent substrate and protein, measuring the signal of the tracking dye (e.g., fluorescence) and normalizing the bioluminescent signal to the signal of the tracking dye. The normalizing may involve finding an average fluorescence signal and determining the deviation of the fluorescence signal in an individual animal from the average fluorescence signal. If the deviation is greater than a set amount (e.g., <NUM>% more or less than the average), the animal is re-imaged. In certain embodiments, the normalizing is done using one or more of Equations (<NUM>), (<NUM>) and (<NUM>), shown below. In certain embodiments, normalizing is done by a computer program (e.g., software).

These and other embodiments will readily occur to those of skill in the art in view of the disclosure herein.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, and recombinant DNA techniques, within the skill of the art. Such techniques are explained fully in the literature. See, e.g.,<NPL>on); <NPL>);<NPL>); <NPL>);<NPL>); and <NPL>).

In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a nucleic acid" includes a mixture of two or more such nucleic acids, and the like.

As used herein, "luminescence" refers to the detectable electromagnetic (EM) radiation, generally, UV, IR or visible light that is produced when the excited product of an exergic chemical process reverts to its ground state with the emission of light. Chemiluminescence is luminescence that results from a chemical reaction. Bioluminescence is chemiluminescence that results from a chemical reaction using biological molecules (or synthetic versions or analogs thereof) as substrates and/or enzymes. Thus, "bioluminescence" refers to the emission of light by biological molecules, particularly proteins. The essential condition for bioluminescence is molecular oxygen, either bound or free in the presence of an oxygenase, a luciferase and ATP, which acts on a substrate, a luciferin. Bioluminescence is generated by an enzyme or other protein (e.g., luciferase) that is an oxygenase that acts on a substrate (e.g., luciferin) and transforms the substrate to an excited state, which upon return to a lower energy level releases the energy in the form of light. Substrates and enzymes for producing bioluminescence, include, for example, luciferin and luciferase, respectively. The luciferin and luciferases may be from any species.

"Luciferase," unless stated otherwise, includes prokaryotic and eukaryotic luciferases, as well as variants possessing varied or altered optical properties, such as luciferases that produce different colors of light (e.g., <NPL>).

"Luciferin" refers to the substrate for luciferase. Luciferin is a low molecular weight organic compound that consists of a benzothiazole moiety attached to a thiazole carboxylic acid moiety. Luciferin is found in fireflies and other animals which, in the presence of ATP and the enzyme luciferase, becomes luminescent. Luciferin is able to pass the blood brain barrier, the blood placenta barrier and the blood testis barrier to distribute quickly through the animal and, toxicity appears low. Luciferin distributes quickly and easily throughout the animal. Luciferin does not affect the animals deleteriously (no evidence of toxicological or immunological effects).

"Light-generating" is defined as capable of generating light through a chemical reaction or through the absorption of radiation.

A "light generating protein" or "light-emitting protein" is a protein capable of generating light. Typically, the light is in the visible spectrum (between approximately <NUM> and <NUM>). Examples include bioluminescent proteins such as luciferases, e.g., bacterial and firefly luciferases.

"Animal" as used herein refers to a non-human mammal, including, without limitation, farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered.

A "transgenic animal" refers to a genetically engineered animal or offspring of genetically engineered animals. A transgenic animal usually contains material from at least one unrelated organism, such as from a virus, plant, or other animal. The "non-human animals" of the invention include vertebrates such as rodents, non-human primates, sheep, dogs, cows, amphibians, birds, fish, insects, reptiles, etc. The term "chimeric animal" is used to refer to animals in which the heterologous gene is found, or in which the heterologous gene is expressed in some but not all cells of the animal.

As used herein, the term "tracking dye" refers to any molecule that, when injected with the bioluminescent substrate, distributes throughout the animal in the same or similar manner as the bioluminescent substrate. Non-limiting examples of "tracking dyes" include, but not limited to, radioactive isotopes, fluorescent molecules, chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, semiconductor nanoparticles, dyes, metal ions, metal sols, and the like. The term "fluorescer" refers to a substance or a portion thereof which is capable of exhibiting fluorescence in the detectable range.

Before describing the compositions and methods in detail, it is to be understood that the disclosure is not limited to particular formulations or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Although a number of methods and materials similar or equivalent to those described herein can be used, exemplary preferred materials and methods are described herein.

The present disclosure relates to compositions (e.g., reagents) comprising a bioluminescent substrate and one or more tracking dyes. These reagents are formulated for injection into a live animal such that the tracking dye(s) and substrate are similarly distributed in the animal. Thus, the signal from the tracking dye(s) can be measured in tissues that provide a luminescent signal and the signal from the tracking dye used to audit and normalize the bioluminescent signal resulting from the reaction of substrate in the reagent and luciferase in the animal. Normalizing the tracking dye signal to the bioluminescent signal allows for greater statistical validity of the bioluminescent measurements.

In addition, the compositions and methods described herein can be used to validate injection of the bioluminescent substrate into the desired target within the organism. For example, if the bioluminescent substrate is intended to be injected into the abdominal cavity so as to be distributed over the animal via the bloodstream, but is mistakenly injected into the intestine, the presence of the tracking dye will allow the researcher to confirm that the substrate is not trapped in an undesired location of the animal.

The compositions (reagents) described herein include both a substrate for a bioluminescent protein and a tracking dye.

Bioluminescent substrates and formulations comprising these substrates are well known in the art and are commercially available. In certain embodiments, the bioluminescent substrate of the invention comprises luciferin (e.g., D-luciferin). For the reagents described herein, the luciferin is typically provided as a potassium salt of defined weight. Liquid reagents may also be used. In preferred embodiments, fresh stocks of luciferin-containing reagents are prepared in the appropriate buffer (e.g., DPBS) just prior to imaging of the animal and the stocks filter sterilized, for example through a <NUM> filter.

Dry luciferin can be reconstituted at the desired concentration, typically from <NUM> to <NUM>/ml. In certain embodiments, the luciferin is reconstituted such that the stock reagent is at <NUM>/ml. Dosages can be readily determined by the skilled artisan. Generally, for administration, luciferin is administered at a dose of approximately <NUM>/kg. Thus, for a <NUM> animal, <NUM>µl of a <NUM>/ml luciferin solution should be administered for delivery of <NUM> of luciferin to the animal.

Luciferin-containing reagents can be administered to live animals by any suitable method, including but not limited to, by intravenous, subcutaneous, intraperitoneal, mucosal routes and the like. Typically, in live animals, luciferin is administered intraperitoneally, optionally with anesthesia. Luciferin kinetic studies can be performed following the instructions provided by the manufacturer (e.g., Caliper Life Sciences).

As noted above, the compositions described herein also comprise a tracking dye. Any tracking dye that is non-toxic and distributes within the animal with the bioluminescent substrate (e.g., luciferin) can be used. In the invention, the tracking dye is a fluorophore. Fluorescent dyes are well known in the art, and include, but are not limited to <NUM>-FAM (Fluorescein) (emits green), Cy <NUM> (emits red), Cy <NUM> (emits purple), Cy <NUM> (emits violet), Cy <NUM> (emits blue), Cy <NUM> (emits near IR), IndoCyanine Green, DyLight <NUM> (emits violet), DyLight <NUM> (emits violet), DyLight <NUM> (emits green), DyLight <NUM> (emits yellow), DyLight <NUM> (emits orange), DyLight <NUM> (emits red), DyLight <NUM> (emits red), DyLight <NUM> (emits far-red), DyLight <NUM> (emits near-IR), DyLight <NUM> (emits near-IR) Alexa Fluor <NUM> (emits cyan-green), Alexa Fluor <NUM> (emits orange), Alexa Fluor <NUM> (emits red), CF <NUM> (emits green), CF <NUM> (emits orange), CF <NUM> (emits red). The excitation maximums and emission maximums of the selected fluorescent dyes are well known and it will be apparent that the tracking dye is selected to emit at a different wavelength than the bioluminescent protein so as to differentiate between the bioluminescent signal and the signal from the tracking dye.

The tracking dye can be added to the luciferin-containing solution prior to or after the luciferin. Thus, the tracking dye can be added following reconstitution of luciferin in the appropriate buffer.

It will be apparent that the concentration of tracking dye included in the compositions as described herein will vary according to the selected dye in a range.

Animals treated with the luciferin and tracking dye compositions as described herein are imaged as described in <CIT> and <CIT> and as described in the materials provided by the manufacturer of the IVIS™ imaging systems, Caliper Life Sciences.

In vivo imaging can be performed using the naked eye or any sort for camera (still or video). In certain embodiments, an intensified CCD camera sensitive enough to detect the bioluminescent signal and with wide enough dynamic range to also detect the fluorescent signal is used for imaging. Suitable cameras are known in the art and include, but are not limited to, an integrated imaging system (IVIS™ Imaging System, Caliper Life Sciences) controlled using LivingImage™ software (Caliper Life Sciences).

The reagent containing the bioluminescent substrate (e.g., luciferin) and tracking dye is typically injected into the intraperitoneal cavity at a luciferin dose of <NUM>/kg body weight (<NUM>/ml Luciferin stock) between about <NUM> minute and <NUM> hour prior to imaging, preferably between <NUM> and <NUM> minutes, including about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> minutes prior to imaging. Mice are typically anesthetized (e.g., Nembutal (<NUM>-<NUM>/kg body weight) or in a gas chamber with an isoflurane/oxygen mixture and isoflurane tubing placed on the animals' noses), and placed on the imaging stage under anesthesia. Animals are typically imaged for between about <NUM> and <NUM> minutes on one or more sides for bioluminescence and from <NUM> to <NUM> seconds for fluorescence. When imaging for fluorescence signal an excitation of <NUM> and an emission of <NUM> can be used depending on the dye selected. Both bioluminescence (photon emission) and fluorescence can be quantified using LivingImage™ software (Caliper Life Sciences).

The compositions described herein allow for the normalization of bioluminescent signals and for the real-time validation of proper distribution of the bioluminescent substrate.

In order to normalize values, the bioluminescent signal from the region of interest is measured according to standard protocols previously described. For the fluorescence measurement a region of interest is selected remote from the site of injection in order to determine the systemic distribution of the dye and substrate. The region of interest is quantified according to standard protocols and recorded in efficiency units.

The fluorescent signal from an individual animal can be compared to the average signal measured for a cohort of animals at a particular time point, or the average for a single animal measured at different time points over a longitudinal study.

A fluorescent signal that deviates more than a pre-determined percentage (e.g., between more than <NUM>%, more preferably between about <NUM>%, and even more preferably more than <NUM>%) from the average signal indicates an aberrant substrate injection and the associated bioluminescent measurement should be discarded or repeated. A fluorescent signal that deviates a lesser percentage from the average can indicate injection variability and the percentage deviation used to correct the bioluminescent signal. Normalizing calculations may be performed by a computer or by hand.

Furthermore, the compositions described herein also allow for the validation of distribution of the bioluminescent substrate within the subject animal. In particular, if it is intended that the bioluminescent substrate be distributed via the bloodstream (e.g., by IP injection in the abdomen), the ability to monitor the location of the fluorophore can provide real-time information on injections, for example if the bioluminescent substrate is localized within a body region or part (e.g., intestines).

Below are examples of specific embodiments for carrying out the present disclosure. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way.

Luciferin/dye formulation was prepared as follows. Ten mL of DPBS to <NUM> of DL800 to dissolve the dye, and aliquoted into <NUM> stock (DL800 stock). Ninety-nine mL of DPBS was added to <NUM> grams of D-Luciferin, Firefly, potassium salt. One mL of DL800 stock was added to <NUM> of D-Luciferin to formulate the final Luciferin/dye with fluorescence dye concentration of <NUM>. 7ug/mL and D-Luciferin concentration of <NUM>/mL.

Mice containing orthotopic tumors were injected intraperitoneal (i. ) with 150µL of Luciferin/fluorescent dye working solution. Six minutes post-injection the mice were placed in a clear Plexiglas anesthesia box (<NUM>-<NUM>% isofluorane) of an IVIS™ imaging systems (Caliper Life Sciences) that allows unimpeded visual monitoring of the animals (e.g. visual determination of breathing). The tube that supplies the anesthesia to the box was plumbed to the anesthesia manifold located inside the imaging chamber. After the mice were fully anesthetized, they were transferred from the box to the nose cones attached to the manifold in the imaging chamber, the door was closed, and the "Acquire" button (part of Living ImageR program) on the computer screen was activated.

For bioluminescence imaging, the imaging time was up to five minutes per side (dorsal/ventral), depending on the experiment. When the mice were turned from dorsal to ventral (or vice versa), they could be visibly observed for any changes in vitality. Bioluminescence imaging utilized the Block and Open filters inside the imaging chamber,.

For fluorescence imaging, the imaging time was from <NUM> to <NUM> seconds per side (dorsal/ventral), depending on the experiment. When imaging for fluorescence signal an excitation of <NUM> and an emission of <NUM> was used. This filter pair was selected using the Living ImageR program.

Bioluminescent and fluorescent signals were quantified using the Living ImageR program as follows. For bioluminescence, a region of interest (ROI) is drawn around the area expressing luminescent signal (<FIG>). This signal is recorded as photons/second. For quantification of fluorescence, the region of interest was placed away from the abdominal region where the substrate was i. injected in order to get a better read out of the systemic distribution of the substrate (see, <FIG>). For dorsal images, the ROI was drawn around the scruff area (back of neck) for quantification of the reference fluorescence signal (<FIG>), while for ventral images the ROI was drawn around the thoracic region (<FIG>). Fluorescent signal was recorded in efficiency units.

Results of imaging are shown in Table <NUM> and <FIG>. In <FIG>, the top panels show bioluminescence and the bottom panels show fluorescence. The animal number is provided above each set of panels.

As can be seen, mouse <NUM> did not provide a strong bioluminescent or fluorescent signal, indicative of a poor injection.

The luciferin/dye formulations described herein have the advantage of utilizing Fluorescent signal to normalize the bioluminescent signal. As described in Example <NUM> and shown in <FIG>, bioluminescent imaging showed an animal (mouse <NUM>) that had a poor injection, as shown by the absence of luminescent signal, and this was confirmed when looking at the corresponding fluorescent images, showing mouse <NUM> with a lower level of dye distribution as compared with the other mice.

The average fluorescent signal obtained in Example <NUM> was obtained, with outliers (mouse <NUM>) omitted. Without mouse <NUM>, the average fluorescence signal was calculated to be <NUM>. The fluorescent signal obtained from each individual animal was then compared to the average fluorescent signal and any animal with a <NUM>% or more decrease in fluorescent signal (as compared to the average) is slated for re-imaging. Using the following formula, the % change in Fluorescence signal for each mouse was determined: <MAT>.

Fluorescence signal normalization results as obtained using Equation (<NUM>) are shown in Table <NUM> and <FIG>.

Since mouse <NUM> showed a decrease (-) of <NUM>% change in fluorescent signal as compared to the average Fluorescent signal, this animal would be re-imaged.

For animals that do not need to be re-imaged, the fluorescence (FLI) Normalization Factor can be used to normalize the bioluminescent (BLI) signal, using the following equations: <MAT> <MAT>.

For animals in which the percent change of the fluorescent signal is below <NUM>% (e.g., mice # <NUM>, <NUM>, <NUM> and <NUM>), the fluorescence normalization value is applied to the quantified Bioluminescent signal. Likewise, if the percent change of fluorescent signal is above <NUM>% (e.g., mice # <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>), the normalized value is subtracted from the quantified Bioluminescent signal. Results of the calculations are shown in Table <NUM> and <FIG>.

Claim 1:
A method of normalizing a bioluminescent signal detected in a live non-human mammalian animal comprising a bioluminescent protein, the method comprising:
providing at least one live non-human mammalian animal, the animal comprising:
(i) the bioluminescent protein; and
(ii) a composition comprising a bioluminescent substrate and a fluorescent tracking dye, wherein the bioluminescent substrate and fluorescent tracking dye are individual components, the composition having been administered in solution in a buffer by injection between <NUM> minute and <NUM> hour prior to imaging, such that the bioluminescent substrate and fluorescent tracking dye are distributed similarly in the animal;
wherein the bioluminescent substrate reacts with the bioluminescent protein to generate a bioluminescent signal having a wavelength; wherein the fluorescent tracking dye emits a fluorescence signal of a different wavelength and wherein the bioluminescent substrate is transformed to an excited state in the presence of the bioluminescent protein, oxygen and ATP;
measuring the bioluminescent signal generated by reaction of the bioluminescent substrate and the bioluminescent protein from the at least one animal;
measuring the fluorescence signal of the tracking dye from the at least one animal: and
normalizing the bioluminescent signal to the fluorescence signal of the tracking dye in the at least one animal, wherein normalizing comprises finding an average fluorescence signal and determining a deviation of the fluorescence signal in an individual animal from the average fluorescence signal, whereby if the deviation is greater than about <NUM>% from the average, the animal is re-imaged by discarding and repeating the measurement of bioluminescence.