WO2001032686A2 - Arginine kinase - Google Patents
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- WO2001032686A2 WO2001032686A2 PCT/US2000/041843 US0041843W WO0132686A2 WO 2001032686 A2 WO2001032686 A2 WO 2001032686A2 US 0041843 W US0041843 W US 0041843W WO 0132686 A2 WO0132686 A2 WO 0132686A2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1223—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
Definitions
- the present invention relates in general, to arginine kinase (AK) .
- the invention relates to a method of creating metabolic buffering for ischemic tissues and to a method of monitoring transgene expression. These methods involve the delivery of an AK gene into mammalian tissue, e.g., muscle or nervous tissue.
- Cyclocreatine phosphate is a substrate for CK, and is therefore in flux at physiologic pH (Houser et al, J. Mol . Cell. Cardiol . 27:1065-1073 (1995)). This permits the utilization of cyclocreatine phosphate during contraction, decreasing the effective storage for an ischemic event and increasing levels of intracellular inorganic phosphate (Pi) . Additionally, the creatine kinase enzyme functions sub-optimally during intracellular acidosis (Ellington, J. Exp. Biol. 143:177-194 (1989)), reducing its efficacy in ischemic buffering of ATP.
- Cyclocreatine a synthetic compound, is rapidly cleared from the body and must be administered within two hours of the onset of ischemia, in order to afford protection (Elgebaly et al, J. Pharmacol. Exp. Ther. 126:1670- 1677 (1993) ) .
- Vertebrate animals rely on CK to provide buffering of ATP levels in muscle by the following near equilibrium reaction:
- PCr phosphocreatine
- the vertebrate heart does not express the enzyme AK, it does contain levels of the substrate, arginine (approximately 0.5mM) .
- the present invention results, at least in part, from the realization that if AK were introduced into the vertebrate heart, synthesis of 5-8 mM PArg would be anticipated. Given the preferential utilization of PCr during physiological conditions, this additional high energy phosphate backup, would remain until the onset of cellular ischemia.
- the present invention provides a method of establishing a dual source of ATP buffering using gene transfer (e.g., viral gene transfer) . Application of this method results in enhanced protection during ischemia .
- the present invention provides a method that comprises delivering an AK gene (e.g., the gene cloned from Drosophila melanogaster) into mammalian muscle or nervous tissue.
- an AK gene e.g., the gene cloned from Drosophila melanogaster
- Expression of the gene results in the production of the AK protein which generates a pool of arginine phosphate (a high energy phosphate) that can provide metabolic buffering to a tissue during ischemia. This is particularly important in muscular tissues and in the brain.
- the method provides for the creation of a unique signal on phosphorus and proton NMR spectra for both imaging and spectral quantification. This provides a non-invasive signal to report gene transfer when the AK gene is delivered either alone or in conjunction with another gene of interest.
- FIG. 3 Schematic diagram for rAdCMVAK gene construct. Expression was driven by nonspecific cytomegalovirus (CMV) promoter and was stabilized by an SV40 polyadenylation sequence (SV40pA) . From this construct an ⁇ E1- ⁇ E3 adenovirus was prepared using published methods (Hardy et al, J. Virol. 71:1842 (1997) ) .
- CMV nonspecific cytomegalovirus
- SV40pA SV40 polyadenylation sequence
- RT-PCR was utilized to detect the presence of arginine kinase (ArgK) transcripts in rAdCMVAK injected muscles.
- Total RNA isolated from frozen tissue was subjected to reverse transcription and PCR using oligonucleotides specific for drosophila ArgK and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) .
- ArgK transcripts were detected in rAdCMVAK injected muscles ⁇ +AdAK) but not in the contralateral conrol (No Inj ) .
- ArgK and GAPDH primers served as a positive controls for the procedure [ Pos Ctrl ) .
- the model solution consisted of 30 mM PCr and 10 mM Pi and 30 mM PArg in 0.4 mL .
- PArg was well resolved from PCr and ⁇ ATP with a chemical shift of 0.47 ppm relative to PCr.
- the model solution was used to determine the appropriate spectral analysis package for the analysis of both phantom and mouse skeletal muscle data with the minimal amount of user bias.
- the solid line is the best fit to the free induction decay using a Hankel single value decomposition algorithm (van den Boogaart et al, NMR Biomed.
- Figure 7 Stack plot of changes in high energy phosphates during prolonged ischemia in the rAdCMVAK injected limb. During circulatory occlusion, PCr levels are depleted and inorganic Pi levels rise within 15 min of ischemia, followed by the depletion of PArg.
- the present invention relates, generally, to therapeutic and detection methods that comprise the delivery of an AK encoding sequence into mammalian tissue.
- the invention provides a gene transfer approach to creating a dual ATP buffering system in mammalian tissue subject to ischemic stress.
- the dual system: CK for aerobic function and AK for ischemic stress is an effective means to optimize tissue protection.
- the invention provides a highly specific non-invasive method of assessing transgene expression, for example, by phosphorus NMR and proton NMR. This method uses an arginine phosphate signal resulting from delivery of an AK encoding sequence .
- Both aspects of the invention involve the delivery to mammalian tissue of an expression construct that comprises a nucleic acid sequence that encodes AK, or portion thereof that catalyzes the reversible phosphorylation of arginine by ATP.
- Suitable encoding sequences include AK gene sequences cloned from any organism that produces the enzyme of a variety of sources, including Drosophila (e.g., Drosophila melanogaster, or other invertebrate) .
- cDNA sequences can also be used, as can chemically synthesized sequences.
- the construct is structured such that when introduced into target cells, expression of the encoding sequence and production of AK, or portion thereof, result.
- a method that: (1) directs the encoding sequence into specific target cell or tissue types (e.g., muscle (heart, skeletal muscle, smooth muscle) , nervous tissue, kidney, liver) , (2) is efficient in mediating uptake of the construct into the target cell population, and (3) is suited for use in vivo for application.
- specific target cell or tissue types e.g., muscle (heart, skeletal muscle, smooth muscle) , nervous tissue, kidney, liver
- AK encoding sequence (or portion thereof encoding a catalytically active peptide) can be effected using any of a variety of methodologies. Examples include transfection with a viral vector, fusion with a lipid, cationic supported introduction, naked DNA with or without electroporation, DNA conjugates, etc. The selection of which technique to use depends upon the particular situation and its demands. Viral vectors suitable for use include adenoviral vectors, adenoassociated viruses and coxseci .
- Another gene transfer method suitable for use include physical transfer of plasmid DNA in liposomes directly into cells of target tissue. Liposome-mediated DNA transfer has been described by various investigators (Wang and Huang, Biochem. Biphys. Res. Commun. 147:980 (1987); Wang and Huang, Biochemistry 28:9508 (1989); Litzinger and Huang, Biochem. Biophys . Acta 1113:201 (1992); Gao and Huang, Biochem. Biophys. Res. Commun. 179:280 (1991); Feigner, WO 91/17424; WO 91/16024).
- Immunoliposomes can also be used as as carriers of exogenous polynucleotides (Wang and Huang, Proc . Natl. Acad. Sci . USA 84:7851 (1987); Trubetskoy et al, Biochem. Biophys. Acta 1131:311 (1992)). Immunoliposomes can be expected to have improved cell type specificity as compared to liposomes due to the inclusion of specific antibodies that bind to surface antigens on target cell types.
- PL Low molecular weight polylysine
- other polycations can also be used to effect transfection of the construct into cells.
- Zhou et al, Biochem. Biophys. Acta 1065:8 (1991) have reported synthesis of a polylysine-phospholipid conjugate, a lipopolylysine comprising PL linked to N-glutarylphosphatidylethanolamine, which reportedly increases the transfection efficiency of DNA as compared to lipofectin, a commercially used transfection reagent.
- any suitable nucleic acid delivery method can be used in the context of the present invention, including direct physical application of naked DNA comprising the expression construct/transgene to cells of the target tissue population (e.g., via electroporation of naked DNA).
- Expression construct-containing compositions of the invention can be stored and administered in a sterile physiologically acceptable carrier, where the construct is dispersed in conjunction with any agents that aid in the introduction of the constructs into cells.
- a sterile physiologically acceptable carrier including water, PBS, ethanol, lipids, etc.
- concentration of the construct which will be sufficient to provide a therapeutic effect or to give rise to a detectable signal will depend on the efficiency of transport into the cells.
- Actual delivery of the construct, formulated as described above, can be carried out by a variety of techniques including direct injection, intravenous injection and other physical methods (including microprojectiles to target visible and accessible regions of tissue (e.g., with naked DNA) .
- Administration may be by syringe needle, cannula, catheter, etc, as a bolus, a plurality of doses or extended infusion, etc.
- compositions containing the present constructs can be administered for therapeutic and/or detection purposes.
- compositions can be administered to a patient at risk of the effects of ischemic stress (e.g., patients undergoing cardiac surgery (for any type of repair, including vascular manipulations) , patient at risk of ischemic attack due to cardiomyopathy or vascular problems) .
- compositions can also be administered to potential heart donors prior to organ harvest. Amounts effective for this use will depend upon the severity of the condition, the general state of the patient, and the route of administration. In the case of detection, the composition can be administered to a patient undergoing gene therapy. Amounts effective for this use will depend on the patient and route of administration .
- the expression construct of the invention is introduced into mammalian skeletal tissue to provide an additional cytoplasmic ATP buffer.
- the CK system is poised to keep the ATP/ADP at very high levels, enabling high ATPase fluxes at the onset of burst activity at the cost of PCr.
- PCr cannot continue to sustain its thermodynamic buffer capacity at low ATP/ADP ratios which exist during sustained activity or prolonged ischemia. Ultimately, ATP levels are depleted, rigor ensues, and Ca ⁇ accumulation occurs in the sarcoplasm resulting in the loss of membrane integrity and cell death. Based on the difference in the CK and AK equilibrium constants, if the two enzymes exist in the same cell, then the initial flux through the AK reaction is small compared to that through CK (Ellington, J. Exp. Biol . 143:177- 194 (1989) ) . As PCr is depleted, the ATPase flux is primarily supported by AK.
- AK and CK are beneficial to skeletal cells under conditions of prolonged ischemia or fatiguing conditions.
- a large PArg pool is formed without disturbing the normal levels of ATP or PCr (Sweeney, Med. Sci . Sports Exerc . 26:30-36 (1994)) .
- PCr which occurs in ischemia
- CK can no longer buffer changes in ATP.
- the PArg pool continues to buffer changes in ATP levels.
- the AK reaction slows the fall of pH.
- AK serves as a noninvasive monitoring system for viral mediated gene delivery in skeletal muscle.
- AK can be used as a marker in any tissue in which free arginine levels are such that the resulting PArg resonance is above the noise limit (e.g., >0.2mM) .
- This transgene upholds a number of criteria necessary for a gene marker in that it is small, nontoxic, and unique against the mammalian background.
- the expression this particular marker in striated muscle introduces an additional thermodynamic buffer into a highly energetic tissue. This can provide insight into the role of phosphagen kinases in cellular function.
- MRS magnetic resonance spectroscopy
- the present invention provides the first MRS method capable of directly monitoring the expression of a unique, nontoxic gene marker in vivo .
- This method has the advantage that when tissue specificity or when inducible expression is desired, the reporter and therapeutic gene are under control of the same promoter. Furthermore this method is not hampered by indicator delivery limitations . Certain aspects of the invention are described in greater detail in the non-limiting Examples that follow.
- the cDNA encoding for AK was isolated from an adult Drosophilia melangaster cDNA library via the Polymerase Chain Reaction (PCR) .
- the following primers were designed (Sense strand: CGCCCTTTTACAATGGTCAAT and Antisense Strand: GATTGTTGCGTATGCCCCGAA) .
- An adenovirus transfer plasmid (pAdLox) was constructed that contained a Cytomegalovirus (CMV) promoter, the AK cDNA, simian virus 40 polyadenylation signal, and the genetic material necessary for viral packaging.
- Fig. 3 shows a schematic for this gene construct.
- Adenovirus was prepared by the University of North Carolina Gene Therapy Vector Core (Chapel Hill, NC) , following published procedures (Snyder et al, In Current Protocols in Human Genetics, eds. Dracopoli and Haines (1996) ) .
- New Zealand White Rabbits (3 -5kg, 10-15 weeks old) were used in the study. Each animal was fully anesthetized and mechanically ventilated. The animal was prepped and draped in a sterile fashion. A left thoractomy was performed through the fourth intercostal interspace. The pericardium was opened and the lateral branch of the left coronary artery (LCA) was identified. 200 ⁇ l of 10% glycerol/PBS containing 5xl0 10 adenoviral particles were directly injected in five 40 ⁇ l aliquots throughout the distribution. The chest was closed and the animal recovered without further intervention for seven days .
- LCA left coronary artery
- the animal was then returned to the operating room for non-survival surgery.
- a repeat left thoracotomy was performed.
- Two piezoelectric sononmicrocrystals (Sonometrics Corp) were placed in the region previously injected.
- a 7-0 Prolene (Ethicon) suture was placed around the lateral branch of the LCA, just proximal to the region injected.
- a red rubber catheter was used to occlude the artery for 30 minutes. Measurements were obtained at baseline, 15, and 30 minutes of occlusion. Subsequently, the ligature was released and reperfusion was measured for 60 minutes. Measurements were obtained at 2 , 15, 30, 45, and 60 minutes of reperfusion. Control animals underwent identical procedures but without viral injection.
- TTC triphenyltetrazolium
- TTC incubation Analysis of infarct percentage in the area at risk was assessed by TTC incubation, as previously described (Jacobs et al, Science 260:819-822 (1993)) . Images both pre- and post- incubation of TTC were scanned into Adobe Photoshop. IP lab software was used to first quantify the area at risk in the pre-TTC images . The infarct area was then defined by the same method and expressed as a percentage of the area at risk.
- RNA isolated from frozen tissue was subjected to reverse transcription and PCR (Perkin- Elmer) using oligonucleotides specific for Drosophilia AK (CGCCCTTTTACAATGGTCAAT, sense primer; GATTGTTGCGTATGCCCCGAA, antisense primer) .
- Primers that amplified ⁇ Actin (CLONTECH) served as a positive control for the procedure.
- the virus construct was prepared as described in Example 1.
- Injections 40 ⁇ l of 10% glycerol/PBS containing approximately 10-L0 adenoviral particles were injected into the interstitial space of the anterior and posterior muscle compartment of the right hindlimb of anesthetized C57BL/6 neonatal mice between 1 and 5 days of age. Once the mice regained consciousness they were returned to the animal facility until further study.
- RT-PCR Detection of Transgene Expression RT-PCR was utilized to detect the presence of arginine kinase transcripts in rAdCMVAK injected muscles.
- Total RNA isolated from frozen tissue using a commercial kit (RNAqueous, Ambion, Austin, TX) and was subjected to reverse transcription and PCR (Perkin Elmer, CA) using oligonucleotides specific for drosophila AK (TGCCGAGGCTTACACAG, sense primer; AAGTGGTCGTCGATCAG, antisense primer) .
- High resolution NMR spectra were recorded in both the 2wk-8month old hindlimbs using a 5 mm diameter surface coil double tuned to ⁇ H (300 Mhz) and 31 P (121 Mhz) on a Bruker 300 MHz AMX spectrometer. Magnetic field homogeneity was adjusted using the free proton signal, resulting in a typical full width half maximum of 0.2 ppm.
- 31 _ MRS spectra were obtained with a pulse repetition time of 5.4 s, a pulse width of 20 ⁇ seconds, a spectral width of 12,000 Hz and 4096 complex data points. Peak areas, chemical shifts, and line widths were measured using time domain analysis (Fig. 5). All chemical shifts were determined relative to the PCr resonance.
- the extensor digitorum longus (EDL) was removed from the hind limb, retaining the proximal and distal tendon.
- the intact muscle was immersed in a Ringers solution buffered to pH 7.4 with 25mM HEPES, which will be continuously oxygenated and maintained at 25 ⁇ 0.5°C.
- Muscles were mounted horizontally in the muscle bath, attached by tendinuous insertions to a platform at one end and to the lever of a dual mode servomotor system at the other. Muscle length was adjusted to the length (LO) at which maximal twitch force is reached. Stimulation was delivered via two platinum plate electrodes which are positioned along the length of the muscle. The maximal tetanic force was determined using 120 Hz, 1.5sec supramaximal pulses.
- a solution of the expected physiological concentrations of ATP, Pi, PCr and PArg was constructed. As seen in Fig. 5, PArg was well resolved from PCr and ⁇ ATP with a chemical shift of 0.47 ppm relative to PCr. In addition, the solution was used to determine the appropriate spectral analysis package to analyze both phantom and mouse skeletal muscle data with the minimal amount of user bias. Based on these preliminary studies a time domain HSVD algorithm (van den Boogaart et al, NMR Biomed. 8:87-93 (1995)) was used (Fig. 5) and user peak selection was avoided.
- the PArg resonance was observed in rAdCMVAK muscles up to 8 months post gene delivery, indicating persistent expression of AK.
- the chemical shift of PCr relative to gATP was 2.46 ⁇ 0.02 ppm and was not different between experimental groups and it is the same as previously reported in murine muscle (Steeghs et al, Cell 89:93-103 (1997)).
- Both PCr and PArg existed as single well defined Lorentzian resonances with line widths of 44 ⁇ 2.7 Hz and 32 ⁇ 2.0 Hz, respectively.
- the PArg/ ⁇ ATP ratio in the experimental hindlimb was 1.25 ⁇ 0.12 whereas the PCr/ ⁇ ATP was 1.31 ⁇ 0.12.
- PCr-i-PArg/ ⁇ ATP was 2.56 ⁇ 0.16 in the rAdCMVAK injected leg and PCr/ ⁇ ATP was 2.65 ⁇ 0.58 in control limbs .
- perchloric acid extracts of injected and uninjected EDLs were used to determine ATP levels by HPLC (Wiseman et al, Anal. Biochem. 204:383-389 (1992)). Based on the measured ATP value of 8.7 mM in the injected limb and following correction for saturation, PArg and PCr were calculated to be 11.6 ⁇ 0.85 and 13.6 ⁇ 1.1 mM in the rAdCMVAK injected muscles.
- AK activity in skeletal muscle AK activity was determined in vivo by monitoring the decreases in PArg, PCr and ATP resonances during prolonged ischemia (Fig. 7) .
- PArg was completely resynthesized within 5 minutes of the restoration of blood flow. PArg depletion was demonstrated up to eight months in rAdCMVAK muscle.
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Abstract
The present invention relates in general, to arginine kinase (AK). In particular, the invention relates to a method of creating metabolic buffering for ischemic tissues and to a method of monitoring transgene expression. These methods involve the delivery of an AK gene into mammalian tissue, e.g., muscle or nervous tissue.
Description
ARGININE KINASE
This application claims priority from Provisional Application No. 60/163,660, filed November 5, 1999, the entire contents of that application being incorporated herein by reference.
TECHNICAL FIELD
The present invention relates in general, to arginine kinase (AK) . In particular, the invention relates to a method of creating metabolic buffering for ischemic tissues and to a method of monitoring transgene expression. These methods involve the delivery of an AK gene into mammalian tissue, e.g., muscle or nervous tissue.
BACKGROUND
Several studies have indicated that the dietary ingestion of the creatine analog, cyclocreatine (1 carboxy-methyl-2-iminoimidazolidine) imparts to the myocardium the ability to sustain high levels of ATP during ischemia, by providing additional substrate to the creatine kinase (CK) buffering system (Houser et al, J. Mol. Cell. Cardiol . 27:1065-1073 (1995), Walker, Guanidino Compounds in Biology and Medicine, Chapter 27, pages 187-194 (1987), Turner and Walker, J. Biol. Chem. 262:6605-6609 (1987)). Further studies confirmed that cyclophosphate treated dogs demonstrated restoration of contractility during reperfusion and exhibited less morphologic indices of cell damage in ischemic areas, compared to native
controls (Elgebaly et al, J. Pharmacol. Exp. Ther . 126:1670-1677 (1993)). These investigations demonstrate that ATP levels can be buffered during periods of diminished oxidative phosphorylation, by utilizing an additional intracellular supply of high energy phosphate .
Cyclocreatine phosphate, however, is a substrate for CK, and is therefore in flux at physiologic pH (Houser et al, J. Mol . Cell. Cardiol . 27:1065-1073 (1995)). This permits the utilization of cyclocreatine phosphate during contraction, decreasing the effective storage for an ischemic event and increasing levels of intracellular inorganic phosphate (Pi) . Additionally, the creatine kinase enzyme functions sub-optimally during intracellular acidosis (Ellington, J. Exp. Biol. 143:177-194 (1989)), reducing its efficacy in ischemic buffering of ATP. Cyclocreatine, a synthetic compound, is rapidly cleared from the body and must be administered within two hours of the onset of ischemia, in order to afford protection (Elgebaly et al, J. Pharmacol. Exp. Ther. 126:1670- 1677 (1993) ) .
Vertebrate animals rely on CK to provide buffering of ATP levels in muscle by the following near equilibrium reaction:
MgATP + Creatine <-> Phosphocreatine + MgATP + HA AK is an enzyme found in invertebrates (Storey, Arch. Biochem. Biophys . 179:518-526 (1977) and catalyzes the following reversible reaction:
MgATP + Arginine -» Arginine phosphate + MgADP + HA Evolution has favored CK, likely because it offers rapid buffering ATP concentration at vertebrate physiologic pH and temperature (apparent equilibrium constant is approximately Kck = 100 and Kak = 10 at pH 7.4), and maintains a higher ATP/ADP ratio than other phosphagens . In the heart, CK provides buffering of ATP levels, during each contraction and during transient ischemia. With prolonged ischemia, the phosphocreatine (PCr) pool is depleted preventing efficacious ATP buffering.
While the vertebrate heart does not express the enzyme AK, it does contain levels of the substrate, arginine (approximately 0.5mM) . The present invention results, at least in part, from the realization that if AK were introduced into the vertebrate heart, synthesis of 5-8 mM PArg would be anticipated. Given the preferential utilization of PCr during physiological conditions, this additional high energy phosphate backup, would remain until the onset of cellular ischemia. Accordingly, the present invention provides a method of establishing a dual source of ATP buffering using gene transfer (e.g., viral gene transfer) . Application of this method results in enhanced protection during ischemia .
SUMMARY OF THE INVENTION
The present invention provides a method that comprises delivering an AK gene (e.g., the gene
cloned from Drosophila melanogaster) into mammalian muscle or nervous tissue. Expression of the gene results in the production of the AK protein which generates a pool of arginine phosphate (a high energy phosphate) that can provide metabolic buffering to a tissue during ischemia. This is particularly important in muscular tissues and in the brain. In addition, the method provides for the creation of a unique signal on phosphorus and proton NMR spectra for both imaging and spectral quantification. This provides a non-invasive signal to report gene transfer when the AK gene is delivered either alone or in conjunction with another gene of interest. Objects and advantages of the present invention will be clear from the description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Analysis of Contractility -θ- Native; -so- AK.
Figure 2. AK injection decreases the infarct area/area at risk ratio.
Figure 3. Schematic diagram for rAdCMVAK gene construct. Expression was driven by nonspecific cytomegalovirus (CMV) promoter and was stabilized by an SV40 polyadenylation sequence (SV40pA) . From this construct an ΔE1-ΔE3 adenovirus was prepared using
published methods (Hardy et al, J. Virol. 71:1842 (1997) ) .
Figure 4. RT-PCR was utilized to detect the presence of arginine kinase (ArgK) transcripts in rAdCMVAK injected muscles. Total RNA isolated from frozen tissue was subjected to reverse transcription and PCR using oligonucleotides specific for drosophila ArgK and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) . ArgK transcripts were detected in rAdCMVAK injected muscles { +AdAK) but not in the contralateral conrol (No Inj ) . ArgK and GAPDH primers served as a positive controls for the procedure [ Pos Ctrl ) .
Figure 5. 31p_][Rs spectra of a model solution containing expected physiological concentrations of ATP, Pi, PCr and PArg. The model solution consisted of 30 mM PCr and 10 mM Pi and 30 mM PArg in 0.4 mL . In solution, PArg was well resolved from PCr and γATP with a chemical shift of 0.47 ppm relative to PCr. The model solution was used to determine the appropriate spectral analysis package for the analysis of both phantom and mouse skeletal muscle data with the minimal amount of user bias. The solid line is the best fit to the free induction decay using a Hankel single value decomposition algorithm (van den Boogaart et al, NMR Biomed. 8:87 (1995)) which avoided peak selection and the use of prior knowledge.
Figure 6. In vivo basal 31P spectra from a 6 month old mouse hindlimbs. 3 IP-MRS spectra from the rAdCMVAK injected limb (upper spectrum) reveal a 3IP resonance at the chemical shift for PArg which is not present in the contralateral control limb { lower spectrum) .
Figure 7. Stack plot of changes in high energy phosphates during prolonged ischemia in the rAdCMVAK injected limb. During circulatory occlusion, PCr levels are depleted and inorganic Pi levels rise within 15 min of ischemia, followed by the depletion of PArg.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates, generally, to therapeutic and detection methods that comprise the delivery of an AK encoding sequence into mammalian tissue. In one aspect, the invention provides a gene transfer approach to creating a dual ATP buffering system in mammalian tissue subject to ischemic stress. The dual system: CK for aerobic function and AK for ischemic stress, is an effective means to optimize tissue protection. In a second aspect, the invention provides a highly specific non-invasive method of assessing transgene expression, for example, by phosphorus NMR and proton NMR. This method uses an arginine phosphate signal resulting from delivery of an AK encoding sequence .
Both aspects of the invention involve the delivery to mammalian tissue of an expression construct that comprises a nucleic acid sequence that encodes AK, or portion thereof that catalyzes the reversible phosphorylation of arginine by ATP. Suitable encoding sequences include AK gene sequences cloned from any organism that produces the enzyme of a variety of sources, including Drosophila (e.g., Drosophila melanogaster, or other invertebrate) . cDNA sequences can also be used, as can chemically synthesized sequences. The construct is structured such that when introduced into target cells, expression of the encoding sequence and production of AK, or portion thereof, result. For gene transfer to be practical, it is desirable to employ a method that: (1) directs the encoding sequence into specific target cell or tissue types (e.g., muscle (heart, skeletal muscle, smooth muscle) , nervous tissue, kidney, liver) , (2) is efficient in mediating uptake of the construct into the target cell population, and (3) is suited for use in vivo for application.
Delivery of an AK encoding sequence (or portion thereof encoding a catalytically active peptide) can be effected using any of a variety of methodologies. Examples include transfection with a viral vector, fusion with a lipid, cationic supported introduction, naked DNA with or without electroporation, DNA conjugates, etc. The selection of which technique to use depends upon the particular situation and its demands.
Viral vectors suitable for use include adenoviral vectors, adenoassociated viruses and coxseci .
Another gene transfer method suitable for use include physical transfer of plasmid DNA in liposomes directly into cells of target tissue. Liposome-mediated DNA transfer has been described by various investigators (Wang and Huang, Biochem. Biphys. Res. Commun. 147:980 (1987); Wang and Huang, Biochemistry 28:9508 (1989); Litzinger and Huang, Biochem. Biophys . Acta 1113:201 (1992); Gao and Huang, Biochem. Biophys. Res. Commun. 179:280 (1991); Feigner, WO 91/17424; WO 91/16024).
Immunoliposomes can also be used as as carriers of exogenous polynucleotides (Wang and Huang, Proc . Natl. Acad. Sci . USA 84:7851 (1987); Trubetskoy et al, Biochem. Biophys. Acta 1131:311 (1992)). Immunoliposomes can be expected to have improved cell type specificity as compared to liposomes due to the inclusion of specific antibodies that bind to surface antigens on target cell types.
Low molecular weight polylysine ("PL") and other polycations can also be used to effect transfection of the construct into cells. Zhou et al, Biochem. Biophys. Acta 1065:8 (1991) have reported synthesis of a polylysine-phospholipid conjugate, a lipopolylysine comprising PL linked to N-glutarylphosphatidylethanolamine, which reportedly increases the transfection efficiency of DNA as compared to lipofectin, a commercially used transfection reagent.
Essentially, any suitable nucleic acid delivery method can be used in the context of the present invention, including direct physical application of naked DNA comprising the expression construct/transgene to cells of the target tissue population (e.g., via electroporation of naked DNA). Expression construct-containing compositions of the invention can be stored and administered in a sterile physiologically acceptable carrier, where the construct is dispersed in conjunction with any agents that aid in the introduction of the constructs into cells. Various sterile solutions may be used for administration of the composition, including water, PBS, ethanol, lipids, etc. The concentration of the construct, which will be sufficient to provide a therapeutic effect or to give rise to a detectable signal will depend on the efficiency of transport into the cells.
Actual delivery of the construct, formulated as described above, can be carried out by a variety of techniques including direct injection, intravenous injection and other physical methods (including microprojectiles to target visible and accessible regions of tissue (e.g., with naked DNA) . Administration may be by syringe needle, cannula, catheter, etc, as a bolus, a plurality of doses or extended infusion, etc.
As indicated above, the compositions containing the present constructs can be administered for therapeutic and/or detection purposes. In therapeutic applications, compositions can be
administered to a patient at risk of the effects of ischemic stress (e.g., patients undergoing cardiac surgery (for any type of repair, including vascular manipulations) , patient at risk of ischemic attack due to cardiomyopathy or vascular problems) .
Compositions can also be administered to potential heart donors prior to organ harvest. Amounts effective for this use will depend upon the severity of the condition, the general state of the patient, and the route of administration. In the case of detection, the composition can be administered to a patient undergoing gene therapy. Amounts effective for this use will depend on the patient and route of administration . In a specific embodiment of the first aspect of the invention, the expression construct of the invention is introduced into mammalian skeletal tissue to provide an additional cytoplasmic ATP buffer. The CK system is poised to keep the ATP/ADP at very high levels, enabling high ATPase fluxes at the onset of burst activity at the cost of PCr. PCr, however, cannot continue to sustain its thermodynamic buffer capacity at low ATP/ADP ratios which exist during sustained activity or prolonged ischemia. Ultimately, ATP levels are depleted, rigor ensues, and Ca^÷ accumulation occurs in the sarcoplasm resulting in the loss of membrane integrity and cell death. Based on the difference in the CK and AK equilibrium constants, if the two enzymes exist in the same cell, then the initial
flux through the AK reaction is small compared to that through CK (Ellington, J. Exp. Biol . 143:177- 194 (1989) ) . As PCr is depleted, the ATPase flux is primarily supported by AK. Thus the coexpression of AK and CK is beneficial to skeletal cells under conditions of prolonged ischemia or fatiguing conditions. Following the introduction of AK into the mammalian muscle cytoplasm a large PArg pool is formed without disturbing the normal levels of ATP or PCr (Sweeney, Med. Sci . Sports Exerc . 26:30-36 (1994)) . Upon depletion of PCr, which occurs in ischemia, CK can no longer buffer changes in ATP. At this point, the PArg pool continues to buffer changes in ATP levels. In addition, the AK reaction slows the fall of pH.
In a specific embodiment of the second aspect of the invention, AK serves as a noninvasive monitoring system for viral mediated gene delivery in skeletal muscle. In fact, however, AK can be used as a marker in any tissue in which free arginine levels are such that the resulting PArg resonance is above the noise limit (e.g., >0.2mM) . This transgene upholds a number of criteria necessary for a gene marker in that it is small, nontoxic, and unique against the mammalian background. In addition, the expression this particular marker in striated muscle introduces an additional thermodynamic buffer into a highly energetic tissue. This can provide insight into the role of phosphagen kinases in cellular function.
The use of a magnetic resonance spectroscopy (MRS) visible marker has the obvious clinical benefit of monitoring transgene expression without removing the tissue from the subject. Adenoviruses are known to infect non-replicating cell types such as terminal differentiated myocytes . Muscle has been shown to be an easy target for adenoviral gene transfer in vivo, rendering muscle a viable tissue for gene therapy and the endogenous production of therapeutic secreted proteins (Barton-Davis et al, Proc. Natl. Acad. Sci . USA 95:15603-15607 (1998), Rivard et al, Am. J. Pathol. 154:355-363 (1999), Gao et al, Hum. Gene. Ther. 9:2353-2362 (1998)). With the advent of high titer recombinant adeno- associated viruses (AAV) which lack immunogenic response, human gene therapy using AAV has become a reality (Barton-Davis et al, Proc. Natl. Acad. Sci. USA 95:15603-15607 (1998), Haecker et al, Hum. Gene Ther. 7:1907-1914 (1996), Gao et al, Hum. Gene. Ther. 9:2353-2362 (1998)). Due to the large volume of tissues necessary to target in humans, methods are need to assess the degree of gene transfer achieved. Moreover, due to the possibility that only transient expression (Ye et al, Science 283:88- 91 (1999)) of the therapeutic gene is achieved, this method should be non-damaging, nontoxic, and capable of sequential measures. Whereas attempts to use MRI to monitor gene expression have relied on receptor mediated or probe activation strategies (Bogdanov and Weissleder, Trends Biotechnol . 16:5-10 (1998), Moore et al, Biochim. Biophys. Acta 1402:239-249
(1998)), the present invention provides the first MRS method capable of directly monitoring the expression of a unique, nontoxic gene marker in vivo . This method has the advantage that when tissue specificity or when inducible expression is desired, the reporter and therapeutic gene are under control of the same promoter. Furthermore this method is not hampered by indicator delivery limitations . Certain aspects of the invention are described in greater detail in the non-limiting Examples that follow.
EXAMPLE 1
Viral Gene Transfer of AK Provides Functional Myocardial ATP Buffering During Ischemia
Experimental Details
Viral construction
The cDNA encoding for AK was isolated from an adult Drosophilia melangaster cDNA library via the Polymerase Chain Reaction (PCR) . The following primers were designed (Sense strand: CGCCCTTTTACAATGGTCAAT and Antisense Strand: GATTGTTGCGTATGCCCCGAA) . An adenovirus transfer plasmid (pAdLox) was constructed that contained a Cytomegalovirus (CMV) promoter, the AK cDNA, simian virus 40 polyadenylation signal, and the genetic material necessary for viral packaging. Fig. 3
shows a schematic for this gene construct. Adenovirus was prepared by the University of North Carolina Gene Therapy Vector Core (Chapel Hill, NC) , following published procedures (Snyder et al, In Current Protocols in Human Genetics, eds. Dracopoli and Haines (1996) ) .
Animal surgery
New Zealand White Rabbits (3 -5kg, 10-15 weeks old) were used in the study. Each animal was fully anesthetized and mechanically ventilated. The animal was prepped and draped in a sterile fashion. A left thoractomy was performed through the fourth intercostal interspace. The pericardium was opened and the lateral branch of the left coronary artery (LCA) was identified. 200μl of 10% glycerol/PBS containing 5xl010 adenoviral particles were directly injected in five 40 μl aliquots throughout the distribution. The chest was closed and the animal recovered without further intervention for seven days .
The animal was then returned to the operating room for non-survival surgery. A repeat left thoracotomy was performed. Two piezoelectric sononmicrocrystals (Sonometrics Corp) were placed in the region previously injected. A 7-0 Prolene (Ethicon) suture was placed around the lateral branch of the LCA, just proximal to the region injected. A red rubber catheter was used to occlude the artery for 30 minutes. Measurements were
obtained at baseline, 15, and 30 minutes of occlusion. Subsequently, the ligature was released and reperfusion was measured for 60 minutes. Measurements were obtained at 2 , 15, 30, 45, and 60 minutes of reperfusion. Control animals underwent identical procedures but without viral injection. In the subset of animals undergoing "area at risk" measurements, the ligature was refastened. 5ml of methylene blue (Sigma) was injected into the left atrium. The animal was euthanized and the heart was procured. 5mm sections were prepared and photographs taken. The sections were then incubated in triphenyltetrazolium (TTC) (Jacobs et al, Science 260:819-822 (1993)) at 37 °C for 1 hour.
Functional analysis and assessment of infarct percentage
Functional analysis was studied by crystal sonomicrometry, a technique that measures left ventricle (LV) segmental length changes during each cardiac cycle. Two 1mm piezoelectric crystals were implanted into the myocardial wall, approximately 10mm apart, in the distribution of the lateral branch of the LCA. Regional ischemia was induced as described above. Measurements were recorded at baseline, 15, and 30 minutes of arterial occlusion. Subsequently, the ligature was unfastened and measurements were then recorded at 2 , 15, 30, 45, and 60 minutes of reperfusion. Fractional shortening is defined as the difference between the
end-diastolic length and the end-systolic length divided by the end-diastolic length (the distance between the crystals) . The data was analyzed with Sonoview software (Sonometrics Corp) and reported as a percentage of baseline ± SEM.
Analysis of infarct percentage in the area at risk was assessed by TTC incubation, as previously described (Jacobs et al, Science 260:819-822 (1993)) . Images both pre- and post- incubation of TTC were scanned into Adobe Photoshop. IP lab software was used to first quantify the area at risk in the pre-TTC images . The infarct area was then defined by the same method and expressed as a percentage of the area at risk.
Detection of transgene expression
One week after injection of the viral vector, the hearts were procured, rapidly frozen and stored at -80°C. Two lOOmg aliquots of tissue were obtained from each heart; the first from the injected portion and the second from a distant site. Total RNA isolated from frozen tissue (RNAqueous) was subjected to reverse transcription and PCR (Perkin- Elmer) using oligonucleotides specific for Drosophilia AK (CGCCCTTTTACAATGGTCAAT, sense primer; GATTGTTGCGTATGCCCCGAA, antisense primer) . Primers that amplified β Actin (CLONTECH) served as a positive control for the procedure.
Statistical analysis
All data were expressed as mean value ± standard error of the mean (S.E.M.). The Student's t-test was used to calculate the P value (P<.05) was considered to be significant.
Results
The expression of AK mRNA resulting from direct injection myocardial injection of the recombinant adenovirus was confirmed by RT-PCR. Figure 1 compares functional measurements of regional contractility in the AK group (n=10) to the native group (n=5) . There was no statistical difference in the baseline fractional shortening between the AK group and the native group. The AK group maintained 75.8%±3.0 of baseline contractility at 15minutes of ischemia, compared to 34.7%±1.8 in controls (P<0.0001). At thirty minutes of ischemia, effective contractility was sustained in AK hearts, but persistently decreased in the native hearts (83.7+6.0 vs. 30.5±2.1, p<0.0001). Upon reperfusion, a transient hypercontractility was observed in the native group (30.5%± 2.1 @ the conclusion of ischemia vs. 60.1%±2.4 @ 2min reperfusion; p<0.001). This increase is not observed in the AK group (83.7%±6.0@ 30min ischemia vs. 75.9%±6.2 at 2min reperfusion) . The AK group maintained a significantly higher percentage of baseline contractility at each time point in
reperfusion (p-values = 0.008, 0.002, 0.001, and <0.0001 at 15, 30, 45, and 60 minute of reperfusion, respectively) There was significant decrease in contractility with prolonged reperfusion in the control group ( 60.2%±2.4@2min reperfusion vs.
33.8%±2.9@60min reperfusion; p<0.0005). This change was not observed in the AK group (75.9%±6.2@2min reperfusion vs. 78.4%±3.8@60min reperfusion). Further data demonstrated no difference in contractility at baseline, during ischemia or during reperfusion, between native hearts and those injected with adenovirus containing the reporter gene LacZ (an adenovirus that codes for a gene that does not provide ATP buffering capacity) . AK injection decreased the infarct area/area at risk ratio from 52.08%±1.9 in native hearts (n=4) to 7.21%±1.59 in the AK group (p<0.0001). These data are shown schematically in Figure 2. No significant difference in the infarct area/area at risk ratio in native hearts 52.08%±1.9 compared to hearts injected with adenovirus encoding the reporter gene LacZ (49.5%±4) .
EXAMPLE 2
Noninvasive Measurement of Gene Expression in Skeletal Muscle
Experimental Details
Virus construct
- IS
The virus construct was prepared as described in Example 1.
Injections 40 μl of 10% glycerol/PBS containing approximately 10-L0 adenoviral particles were injected into the interstitial space of the anterior and posterior muscle compartment of the right hindlimb of anesthetized C57BL/6 neonatal mice between 1 and 5 days of age. Once the mice regained consciousness they were returned to the animal facility until further study.
RT-PCR Detection of Transgene Expression RT-PCR was utilized to detect the presence of arginine kinase transcripts in rAdCMVAK injected muscles. Total RNA isolated from frozen tissue using a commercial kit (RNAqueous, Ambion, Austin, TX) and was subjected to reverse transcription and PCR (Perkin Elmer, CA) using oligonucleotides specific for drosophila AK (TGCCGAGGCTTACACAG, sense primer; AAGTGGTCGTCGATCAG, antisense primer) . Primers which amplified glyceraldehyde 3-phosphate dehydrogenase ( GAAGGTCGGAGTCAACGGATTTGGT, sense primer; CATGTGGGCCATGAGGTCCACCAC, antisense primer) served as a positive control for the procedure.
NMR
High resolution NMR spectra were recorded in both the 2wk-8month old hindlimbs using a 5 mm
diameter surface coil double tuned to ^H (300 Mhz) and 31P (121 Mhz) on a Bruker 300 MHz AMX spectrometer. Magnetic field homogeneity was adjusted using the free proton signal, resulting in a typical full width half maximum of 0.2 ppm. 31 _ MRS spectra were obtained with a pulse repetition time of 5.4 s, a pulse width of 20 μseconds, a spectral width of 12,000 Hz and 4096 complex data points. Peak areas, chemical shifts, and line widths were measured using time domain analysis (Fig. 5). All chemical shifts were determined relative to the PCr resonance.
In situ measurements Intracellular pH was determined based on the Pi chemical shift. AK activity was detected in vivo by monitoring the degradation of PArg levels during circulatory occlusion (Fig. 7) . Prolonged ischemia was produced by the application of a tourniquet around the upper thigh muscles.
In Vi tro Force Measurment
The extensor digitorum longus (EDL) was removed from the hind limb, retaining the proximal and distal tendon. The intact muscle was immersed in a Ringers solution buffered to pH 7.4 with 25mM HEPES, which will be continuously oxygenated and maintained at 25±0.5°C. Muscles were mounted horizontally in the muscle bath, attached by tendinuous insertions to a platform at one end and to the lever of a dual
mode servomotor system at the other. Muscle length was adjusted to the length (LO) at which maximal twitch force is reached. Stimulation was delivered via two platinum plate electrodes which are positioned along the length of the muscle. The maximal tetanic force was determined using 120 Hz, 1.5sec supramaximal pulses.
Results
MRS Measurment of AK expression in skeletal muscle
Solution experiments
To determine the feasibility of measuring AK activity in vertebrate muscle, a solution of the expected physiological concentrations of ATP, Pi, PCr and PArg was constructed. As seen in Fig. 5, PArg was well resolved from PCr and γATP with a chemical shift of 0.47 ppm relative to PCr. In addition, the solution was used to determine the appropriate spectral analysis package to analyze both phantom and mouse skeletal muscle data with the minimal amount of user bias. Based on these preliminary studies a time domain HSVD algorithm (van den Boogaart et al, NMR Biomed. 8:87-93 (1995)) was used (Fig. 5) and user peak selection was avoided.
Detection of AK expression in skeletal muscle by NMR
Due to the small volume of muscle in the neonatal mouse hindlimb (1-5 day old) the earliest
possible 3-1-P-MRS measurements were made 12-13 days post gene delivery. At this time point a unique resonance was observed at 0.49 ppm relative to the PCr resonance. This resonance at 0.49±0.01 (n=24) ppm away from the PCr resonance was observable in all of the injected limbs and was not present in the contralateral control leg or vehicle injected limbs (Fig. 5) . The presence of arginine kinase transcripts in rAdCMVAK injected muscles was confirmed by RT-PCR (Fig. 4) . The PArg resonance was observed in rAdCMVAK muscles up to 8 months post gene delivery, indicating persistent expression of AK. The chemical shift of PCr relative to gATP was 2.46±0.02 ppm and was not different between experimental groups and it is the same as previously reported in murine muscle (Steeghs et al, Cell 89:93-103 (1997)). Both PCr and PArg existed as single well defined Lorentzian resonances with line widths of 44±2.7 Hz and 32±2.0 Hz, respectively. The PArg/γATP ratio in the experimental hindlimb was 1.25±0.12 whereas the PCr/γATP was 1.31±0.12. The total PCr-i-PArg/γATP was 2.56±0.16 in the rAdCMVAK injected leg and PCr/γATP was 2.65±0.58 in control limbs . In order to calculate absolute PCr and PArg concentration, perchloric acid extracts of injected and uninjected EDLs were used to determine ATP levels by HPLC (Wiseman et al, Anal. Biochem. 204:383-389 (1992)). Based on the measured ATP value of 8.7 mM in the injected limb and following correction for saturation, PArg and PCr
were calculated to be 11.6±0.85 and 13.6±1.1 mM in the rAdCMVAK injected muscles.
Detection of AK activity in skeletal muscle AK activity was determined in vivo by monitoring the decreases in PArg, PCr and ATP resonances during prolonged ischemia (Fig. 7) . After lhr of ischemia intracellular pH was 6.73±0.06, PCr/ATP decreased by 77±8%, whereas PArg/ATP decreased by 50±15% of basal levels (n=6) . PArg was completely resynthesized within 5 minutes of the restoration of blood flow. PArg depletion was demonstrated up to eight months in rAdCMVAK muscle. These results demonstrate AK activity and the equlibration of the PArg pool with cytoplasmic ATP.
In vitro mechanics
The expression of AK in the EDL did not have a detrimental effect on maximal force production. The EDLs of three, six month old mice were removed for the determination of in vi tro maximal tetanic muscle force was determine. There was no significant difference in maximal tetanic force between rAdCMVAK and the contra lateral control limb. Furthermore, the specific forces were not different from uninjected control EDLs.
All documents cited above are hereby incorporated in their entirety by reference.
One skilled in the art will appreciate from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention.
Claims
1. A method of producing a metabolic buffering system in a mammalian tissue comprising introducing into said tissue arginine kinase under conditions such that production of said buffering system is effected.
2. The method according to claim 1 wherein said tissue is subject to ischemic stress.
3. The method according to claim 1 wherein an expression construct comprising a nucleic acid sequence encoding said arginine kinase, or portion thereof that catalyzes the reversible phosphorylation of arginine by ATP, is introduced under conditions such that said nucleic acid sequence is expressed and said arginine kinase is thereby produced.
4. The method according to claim 3 wherein said arginine kinase is Drosophila melanogaster arginine kinase.
5. The method according to claim 1 wherein said tissue is muscle or nervous tissue.
6. The method according to claim 1 wherein said tissue is present in a patient.
7. The method according to claim 6 wherein said patient is at risk of the effects of ischemic stress or is at risk of ischemic attack.
8. The method according to claim 6 wherein said tissue is heart or brain tissue.
9. The method according to claim 1 wherein creatine kinase is present in said tissue.
10. A method of monitoring for gene delivery to a tissue comprising introducing into said tissue an expression construct comprising a nucleic acid sequence encoding arginine kinase under conditions such that said sequence is expressed and monitoring said tissue for the presence of phosphoarginine .
11. The method according to claim 10 wherein said tissue is mammalian tissue.
12. The method according to claim 10 wherein said tissue is muscle tissue.
13. The method according to claim 10 wherein said construct is present in a viral vector.
14. The method according to claim 10 wherein said monitoring is effected using magnetic resonance spectroscopy .
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Non-Patent Citations (4)
Title |
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DATABASE CAPLUS [Online] JAMES ET AL.: 'Distribution and genetic basis of arginine kinase in wild type and flightless mutants of drosophila melanogaster', XP002940824 Database accession no. 1989:4998 & JOURNAL OF EXPERIMENTAL ZOOLOGY vol. 248, no. 2, 1988, pages 185 - 191 * |
SHOFER ET AL.: 'Effects of hypoxia and toxicant exposure on adenylate energy charge and cytosolic ADP concentrations in abalone' COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY vol. 119C, no. 1, January 1998, pages 51 - 57, XP002940823 * |
WALTER ET AL.: 'Noninvasive measurement of gene expression in skeletal muscle' PROC. NATL. ACAD. SCI. USA vol. 97, no. 10, 09 May 2000, pages 5155 - 5155, XP002940825 * |
WANG ET AL.: 'Arginine kinase expression and localization in growth cone migration' THE JOURNAL OF NEUROSCIENCE vol. 18, no. 3, 01 February 1998, pages 987 - 998, XP002940822 * |
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