(en)The present invention is directed to methods for the treatment or prevention of starvation in a cell, e.g., a neuronal cell, and methods for the treatment and prevention of disorders associated therewith by the administration of an agent, e.g., a nucleic acid molecule, which enhances the intracellular generation and/or uptake of glucose, pyruvate, lactate, and/or NADPH.

1.ApplicationNumber: US-201013264515-A
1.PublishNumber: US-9610363-B2
2.Date Publish: 20170404
3.Inventor: CEPKO CONSTANCE L.
PUNZO CLAUDIO

4.Inventor Harmonized: CEPKO CONSTANCE L(US)
PUNZO CLAUDIO(US)

5.Country: US
6.Claims:
(en)The present invention is directed to methods for the treatment or prevention of starvation in a cell, e.g., a neuronal cell, and methods for the treatment and prevention of disorders associated therewith by the administration of an agent, e.g., a nucleic acid molecule, which enhances the intracellular generation and/or uptake of glucose, pyruvate, lactate, and/or NADPH.

7.Description:
(en)RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 61/169,835, filed on Apr. 16, 2009, the entire contents of which are incorporated herein by this reference.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with Government support under contract EY014466 awarded by the National Institutes of Health. The government has certain rights in the invention.


FIELD OF THE INVENTION
The present invention is directed to methods for the treatment or prevention of starvation in a cell and disorders associated therewith by the administration of an agent which enhances the intracellular generation and/or uptake of glucose and/or intracellular generation and/or uptake of NADPH and/or intracellular generation and/or uptake of pyruvate and/or intracellular generation and/or uptake of lactate.
BACKGROUND OF THE INVENTION
Cells can be compromised by genetic and environmental factors that lead to their malfunction and death. For example, in the retina, specialized sensory neurons, the photoreceptors (rods and cones), as well as ganglion cells, the output neurons of the retina, are the neuronal cell types that can malfunction and die due to genetic and/or environmental reasons, leading to partial or complete loss of vision.
The retina contains two major types of light-sensitive photoreceptor cells, i.e., rod cells and cone cells. Cone cells are responsible for color vision and require brighter light to function, as compared to rod cells. There are three types of cones, maximally sensitive to long-wavelength, medium-wavelength, and short-wavelength light (often referred to as red, green, and blue, respectively, though the sensitivity peaks are not actually at these colors). Cones are mostly concentrated in and near the fovea. Only a small percentage of photoreceptors are cones in the periphery of the retina. Objects are seen most sharply in focus when their images fall on the cone-enriched spot, as when one looks at an object directly. Cone cells and rods are connected through intermediate cells in the retina to nerve fibers of the optic nerve. When rods and cones are stimulated by light, the nerves send off impulses through these fibers to the brain.
Reduced viability of cone cells is associated with various retinal disorders, in particular, retinitis pigmentosa. Retinitis pigmentosa is a family of inherited retinal degenerations (RD) that is currently incurable and frequently leads to blindness. Affecting roughly 1 in 3,000 individuals, it is the most prevalent form of RD caused by a single disease allele (RetNet, www.sph.uth.tmc.edu/Retnet/). The phenotype is characterized by an initial loss of night vision due to the malfunction and death of rod photoreceptors, followed by a progressive loss of cones (Madreperla, S. A., et al. (1990) Arch Ophthalmol 108, 358-61). Additionally, retinitis pigmentosa is further characterized by the following manifestations: night blindness, progressive loss of peripheral vision, eventually leading to total blindness, ophthalmoscopic changes consist in dark mosaic-like retinal pigmentation, attenuation of the retinal vessels, waxy pallor of the optic disc, and in the advanced forms, macular degeneration. Since cones are responsible for color and high acuity vision, it is their loss that leads to a reduction in the quality of life. In many cases, the disease-causing allele is expressed exclusively in rods; nonetheless, cones die too. Indeed, to date there is no known form of RD in humans or mice where rods die, and cones survive. In contrast, mutations in cone-specific genes result only in cone death.
SUMMARY OF THE INVENTION
The present invention is directed to methods for inhibiting starvation of a cell, as well as methods for treating or preventing a disorder associated with starvation of a cell. The present invention is based, at least in part, on the discovery that the upregulation of certain genes in a cell undergoing starvation can serve to enhance intracellular levels of glucose, lactate, pyruvate, and/or NADPH. In particular, the upregulation of genes encoding enzymes involved in glucose transport, glucose production (gluconeogenesis), lactate transport and NADPH production can serve to inhibit cellular starvation, thus increasing cellular viability.
Accordingly, the present invention provides methods for inhibiting starvation of a cell as well as methods for the treatment and/or prevention of disorders associated with cellular starvation, for example, retinitis pigmentosa, by enhancing the intracellular levels of glucose, pyruvate, lactate and/or NADPH.
In one aspect, the present invention is directed to a method for inhibiting starvation of a cell by contacting the cell with an agent that enhances the intracellular generation and/or uptake of glucose and/or lactate and/or pyruvate and/or NADPH in the cell. In another aspect, the present invention is directed to a method for treating or preventing a disorder associated with starvation of a cell in a subject by administering to the subject an agent that enhances the intracellular generation and/or uptake of glucose and/or lactate and/or pyruvate and/or NADPH. In another aspect, the present invention provides a method for treating or preventing retinitis pigmentosa in a subject by administering to the subject an agent that enhances the intracellular generation and/or uptake of glucose and/or lactate and/or pyruvate and/or NADPH. In yet another aspect, the present invention is directed to a method for prolonging the viability of a cone cell by contacting the cell with an agent that enhances the intracellular generation and/or uptake of glucose and/or lactate and/or pyruvate, and/or NADPH, e.g., for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 2 years, about 3 years, about 4 years, about 5 years, about 10 years, about 15, years, about 20 years, about 25 years, about 30 years, about 40 years, about 50 years, about 60 years, about 70 years, and about 80 years. In another aspect, the present invention is directed to a method for prolonging the viability of a rod cell by contacting the cell with an agent that enhances the intracellular generation and/or uptake of glucose and/or lactate and/or pyruvate, and/or NADPH, e.g., for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 2 years, about 3 years, about 4 years, about 5 years, about 10 years, about 15, years, about 20 years, about 25 years, about 30 years, about 40 years, about 50 years, about 60 years, about 70 years, and about 80 years
Cell types suitable for use in the methods of the invention include, for example, any cell type that is undergoing starvation and/or ischemia, e.g., neuronal cells, skeletal muscle cells, pancreatic islet cells, vascular endothelial cells.
In various embodiments of the foregoing aspects of the invention, the agent enhances the intracellular generation of glucose, enhances the uptake of glucose into a cell, enhances the intracellular generation of NADPH, enhances the intracellular uptake of lactate, and/or enhances the intracellular uptake of pyruvate into a cell, in order to increase the level of intermediates for energy production or anabolic reaction such that the metabolic flux in a cell is enhanced through the pentose phosphate pathway, and/or the ability of a cell to generate phospholipids or other anabolic products is enhanced, and/or the ability of a cell to detoxify free oxygen radicals is enhanced.
In one embodiment, the agent for use in the methods of the invention is a nucleic acid molecule, e.g., a nucleic acid molecule which encodes an enzyme selected from the group consisting of a glucose transporter, a gluconeogenic gene, glucose-6-phosphatase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase, lactate transporter and malic enzyme. The nucleic acid molecule may encode an enzyme involved in the pentose phosphate pathway, for example, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, phosphogluconate dehydrogenase, ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, transketolase or transaldolase.
In another embodiment, the cell is contacted with or the subject is administered at least two nucleic acid molecules which enhance the intracellular generation and/or uptake of glucose and/or pyruvate and/or lactate and/or NADPH, for example, at least two nucleic acid molecules selected from the group consisting of the pyruvate carboxylase gene, the phosphoenolpyruvate carboxykinase gene, and the fructose 1,6-bisphosphatase gene.
In yet another embodiment, the nucleic acid molecule is contained within a vector, for example, a retrovirus, an adenovirus, an adenoviral/retroviral chimera, an adeno-associated virus (AAV), a herpes simplex virus I or II, a parvovirus, a reticuloendotheliosis virus, a poliovirus, a papillomavirus, a vaccinia virus and a lentivirus. In a particular embodiment, the vector is an AAV vector, for example, an AAV 2/5 or an AAV 2/8 vector.
In various embodiments, the disorder is a neurodegenerative disorder, for example, a stroke or Alzheimer's Disease. The disorder may also be an ocular disorder, for example, retinitis pigmentosa, age related macular degeneration, cone rod dystrophy, rod cone dystrophy or glaucoma. In yet another embodiment, the disorder is an ocular disorder associated with the decreased viability of cone cells and/or rod cells. In other embodiments, the ocular disorder is a genetic disorder. In still other embodiments, the disorder is a disorder which deprives cells of glucose, e.g., an ischemic disorder, e.g., heart attack, wound, diabetes, Parkinson's disease.
In certain embodiments, the nucleic acid molecule is administered by injection, e.g., an intraocular injection.
Other features and advantages of the invention will be apparent from the following detailed description and claims



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts rod death kinetics in the Rho-KO mutant described in Example 1 as follows: (a-d) Onset of rod death seen by cleaved nuclear envelope protein LaminA (a), Cleaved Caspase3 (b) (arrowheads) as well as TUNEL (c, d) (arrows: dark gray) (light gray in a, b shows nuclear DAPI staining). (d) Shows a retinal flat mount with view onto the photoreceptor layer. (e-h) Progression of rod death determined by the reduction of the ONL as seen by HE staining. (i-q) End phase of rod death assessed by section analysis (i-l) or by retinal flat mounts (m-q). In the Rho-KO the onset of rod death is around PW5 (a) and progresses up to PW25 (l). By PW17 the ONL is reduced to one row of cells (h, j) and in the following 8 weeks the remaining rods die (j-q) as seen by immunofluorescence with an antibody directed against guanine nucleotide protein alpha transducin (Gnat1) on sections of progressively older animals (j-l). (m-q) Retinal flat mounts showing rods visualized by immunofluorescence with an antibody directed against Gnat1. (m) Shows entire retina while (m, o) show higher magnification around the optic nerve head and (p) shows peripheral region. (q) Shows no signal at PW25 where on sections rods were also not detected (l). Age (in postnatal weeks (PW)) is indicated in the panels. Vertical bar in (a-c, e-l) indicates thickness of the ONL.
FIG. 2 depicts rod death kinetics in the PDE-γ-KO described in Example 1 as follows: (a-d) Onset of rod death seen by cleaved caspase 3 (a, b). At P12, misplaced and excess cells in the INL were dying as part of developmental cell death, as seen in a wild-type control (a) (arrowheads) while in the mutant, cells started to die in the ONL, where photoreceptors reside (b) (arrows). The onset of rod death was also seen by immunofluorescence for the cleaved nuclear envelope protein, LaminA (c) (arrows) as well as TUNEL (d) (arrows; light gray in a-d shows nuclear DAPI staining). Progression of rod death was determined by the reduction of the thickness of the ONL, as seen by HE staining (e-h). (i-q) End phase of rod death was assessed by analysis of sections of progressively older animals. (i-m) Rods were visualized by immunofluorescence with α-rhodopsin or by in situ hybridization for rhodopsin (n-q). (i, j) Retinal section at P16 showing peripheral to central region. (i) Same picture as in (j) with nuclear DAPI stain. (k, l) Higher magnification of section in (i) showing peripheral (k) and central (l) region. As rods die in a central to peripheral manner, more rods were present in the periphery than in the center. By P20, the ONL was reduced to 1 row of cells and rods were found mainly in the periphery (compare arrow (periphery) in (n) to arrowhead (central). The remaining rods in the PDE-γ-KO died over 4 weeks (n-q) as seen on sections. By P49 (q) all rods had died in this mutant (o-q: periphery). Age (in postnatal days (P)) is indicated in the panels. Vertical bar in (a-h, k, l) indicates thickness of the ONL. Data for PDE-β−/− are not shown as they are comparable to the PDE-γ-KO and data on the rod death kinetics of this mutant have been presented in an earlier publication (IOVS, 2007, 48 (2): 849-857).
FIG. 3 depicts cone death kinetics described in Example 1 as follows: (a) qRT-PCR analysis for Opn1sw during cone degeneration. Changes are in indicated as the logarithm of the relative concentration over time on the Y-axis while X-axis indicates postnatal weeks. (b-h, j, k, m-o) Show retinal flat mounts. (k) Shows a retinal section. Medium gray shows PNA expression, light gray shows red/green opsin expression (b, j-n) or blue opsin expression (c, d, o). (b-d) Wild type retina at P35. Red/green opsin (b) and PNA (c, d) expression were detected dorsal and ventral while blue opsin (c, d) was detected only ventrally. (e-g, j-o) Analysis in the PDE-β mutant. (e-g) Central to peripheral gradient of PNA and shortening of cone outer segments (OS). At P20, prior to the major cone death phase, there were fewer elongated OS in the center (e) as compared to the periphery. (f) High magnification of a central or peripheral (g) OS from (e). (h) Wild type OS (white line in f-h marks the OS). (i) Quantification of OS length in central and peripheral regions. The data represents an average of 15 measurements on 3 different retinae of 3 week old mice. With the shortening of OSs during degeneration, red/green opsin was localized throughout the membrane of the cell body and PNA, which detects an extracellular protein(s), was reduced to a small dot attached to the residual OS (j) (arrow: shows red/green and PNA overlap). (k) High magnification of a cone showing red/green localization at the membrane of the main cell body (arrow). (l) Cross section showing red/green in cell body (arrows; j-l P70). Red/green opsin was detected mainly dorsal (l) during degeneration while PNA (m, n) or blue opsin (o) were not altered (m, n: P21, same scale bar; o: P49).
FIG. 4 depicts rod death kinetics in the P23H mutant described in Example 1 as follows. (a-c) Onset of rod death. As rod death progressed very slowly in this mutant, the upregulation of glial fibrillary acidic protein (GFAP) in Muller glia, which has been described as a hallmark of retinal degeneration, was used in conjunction with the other markers to determine the onset of rod degeneration. As seen by antibody staining against GFAP (a, b) degeneration started around PW10 (b). At PW5, GFAP was only found in the ganglion cell layer where it is normally expressed in astrocytes. Consistent with the upregulation of GFAP at PW10, cells positive for cleaved nuclear envelope protein LaminA (c) were also detected (arrow). However, few cells were seen per section due to the slow progression of rod death. (d-f) Progression of rod death determined by the reduction of the ONL as seen by HE staining. (g) End phase of rod death assessed by immunofluorescence with anti-rhodopsin. Although the ONL was reduced to one row of cells by PW35, no end point of rod death was determined. Rods continued to die slowly and even by PW70, many rods were still present (g). Interestingly, most of the rods at that age were confined to the ventral regions of the retina (see also FIG. 6 ). Age (in postnatal weeks (PW)) is indicated in the panels. Vertical bar in (a-f) indicates thickness of the ONL.
FIGS. 5A-5C depict the kinetics and histological changes that accompany rod and cone death across the 4 animal models of RP.
FIG. 6 depicts dorsal cone death kinetics seen by the immunofluorescence with anti-red/green opsin as described in Example 1 as follows: (a-c) Loss of dorsal cones in the Rho-KO mutant over time as seen by the reduced expression of red-green opsin. (d, e) Loss of dorsal cones in the P23H mutant over time. (f, g) Higher magnification of a double staining with an antibody against red/green opsin (medium gray) and rhodopsin (light gray) showing that most rods that survived up to PW80 were in the ventral regions (g) of the retina whereas the red/green expressing cones were mostly dorsal (f).
FIG. 7 depicts affymetrix microarray analysis as described in Example 1 as follows: (a) Equivalent time points in the 4 different mutants at which the microarray analysis was performed (R: approximately halfway through the major phase of rod death; C0: onset of cone death; C1 & C2 first and second time point during cone death respectively). Time is indicated in postnatal days (P) or postnatal weeks (PW). Cartoons depicting the progression of cone death are shown below the corresponding time points. (b) Distribution in percentage of the 195 genes that were annotated. (c) Distribution in percentage of the 68 genes (34.9%) that are part of metabolism in (b).
FIG. 8 depicts that red/green and blue opsin expression was not affected on the RNA level as described in Example 1 as follows: In situ hybridization for red/green opsin (first two rows) or blue opsin (third and fourth row) on retinal sections. RNA levels for red/green opsin and blue were comparable between ventral regions of mutant (first column), wild type animals treated with rapamycin (last column) or untreated wild type animals (second column).
FIG. 9 depicts p*-mTOR in wild type and degenerating retinae as described in Example 1. All panels show immunofluorescence on retinal flat mounts (photoreceptor side up) with the exception of (b, c, g) which show retinal sections. Dark gray the nuclear DAPI stain. (a-c) p*-mTOR levels in wild type retinae. (a) Dorsal (up) enrichment of p*-mTOR. Higher magnification of dorsal and ventral region is shown to the right showing p*-mTOR in red and cone segments in green as detected by PNA. (b, c) Dorsal retinal sections stained for p*-mTOR (medium gray) and PNA (b) (light gray) or α-β-galactosidase (c) (lightest gray). The β-galactosidase is under the control of the human red/green opsin promoter and is expressed in all cones 48 (see Material & Methods). The insets in (b, c) show higher magnification of the cone segments indicating that the p*-mTOR signal is located in the lower part of the outer segment (OS; IS: inner segment). (d-g) Rapamycin treatment of wild type mice leads to downregulation of red/green opsin ventrally (e) but not dorsally (d) (medium gray). Ventral blue opsin (f) (medium gray) remains unaffected, as does PNA (d-g) (light gray). Rapamycin treatment does also not affect mTOR phosphorylation in wild type (g) (medium gray). (h-m) Reduced levels of dorsal p*-mTOR during photoreceptor degeneration (red signal). (h) Wild type control. (i, j) PDE-β mutant. The reduction starts during rod death at P15 (i) as the OSs (light gray: PNA) start to detach from the retinal pigmented epithelium. (i) By P30 only few cones medium gray: α-β-galactosidase) show high levels of p*-mTOR (dark gray). (k-l) A similar reduction is seen in dorsal cones of the other three mutants (cones marked in light gray by PNA). (k) PDE-γ-KO P35. (l) Rho-KO PW20. (m) P23H PW70.
FIG. 10 depicts the dependence of p*-mTOR levels on the presence of glucose as described in Example 1. Different media conditions were tested (a) during 4 hours of retinal explant culture. After culture, retinae were fixed and stained for p*-mTOR (medium gray), PNA (light gray) and DAPI (dark gray). Retinal flat mounts were imaged (b). Dorsal p*-mTOR was only detected when glucose was present in the media.
FIG. 11 depicts the upregulation of Hif-1α and GLUT1 in cones as described in Example 1. All panels show immunofluorescent staining. Left column (a, d, g, h,) shows retinal flat mounts and right column (b, c, e, f, i, j) retinal sections. Dark gray shows nuclear DAPI staining and light gray shows cones marked with PNA. (a-f) Staining for HIF-1α medium gray). (a) Wild type (PW10) (inset) showing higher magnification. (b, c) Cross sections in wild type (PW10). (c) DAPI overlap of (b). (d-f) During cone degeneration in PDE-β−/− (PW10) increased levels of HIF-1α are found in cones (d, inset). (e, f) Cross sections show that the increase of Hif-1α occurs mainly in cones (arrows point to cones that at this stage are located within the top layer of the inner nuclear layer). (f) DAPI overlap of (e). (g) GLUT1 expression in wild type (PW10) (medium gray). Most of the signal in between the cones reflects expression in rods. (h j) Increased expression of GLUT1 in cones during degeneration seen in flat mounts (h) and sections (i j). (i) Overlap of (j) with PNA.
FIG. 12 depicts the upregulation of Hif-1α and GLUT1 in cones as described in Example 1 All panels show immunofluorescent signals within retinal sections. Dark gray shows nuclear DAPI staining and light gray shows cones marked with PNA. (a-d) Staining for HIF-1α (medium gray). (a) Wild-type at PW10 (see also FIG. 11 a - c ). (b) PDE-γ-KO at PW5. (c) Rho-KO at PW20. (d) P23H at PW70. (e-h) Staining for GLUT1 (medium gray). (e) Wild-type at PW10. (f) PDE-γ-KO at PW5 with PNA overlap. (f′) Same image as (f) without PNA. (g) Rho-KO at PW20. (g′) Same image as (g) without PNA. (h) P23H at PW70. (h′) Same image as (h) without PNA. White dotted line marks border between the ONL and INL.
FIG. 13 depicts the increased levels of LAMP-2 at the lysosomal membrane as described in Example 1 as follows: (a-c) Immunofluorescence on retinal flat mounts where LAMP-2 is shown in light gray, red/green opsin in medium and dark gray shows nuclear DAPI stain. Insets in upper right corner (with box) show enlarged cells (arrow). (a) Wild type retinae at PW5 showing lysosome (small light gray dots) with normal LAMP-2 distribution. Weak red/green opsin signal is detected at the level of the PR nuclei since in wild type it is mainly found in the OSs. (b, c) PDE-β mutant at PW5. (b) Enlarged lysosomes (dots) due to accumulation of LAMP-2 at the lysosomal membrane are seen specifically in cones. (c) Confocal section of same field as in (b) taken at the level of the inner nuclear layer showing levels of LAMP-2 similar to those in wild type (a). (d) qRT-PCR for the 3 different LAMP-2 splice forms showing the relative concentration and the ratios between the PDE-β mutant and wild type.
FIG. 14 depicts a retroviral vector, as described in Example 1, encoding a fusion protein between GFP and LC3 as used to infect the retinae of wild type (a-c) and PDE-β−/− (d-f) mice. Light gray signal shows expression of the fusion protein, medium gray signal shows red/green opsin expression, and dark gray signal shows nuclear DAPI staining. (a-f) Retinal flat mounts at PW10 showed uniform expression of the GFP fusion protein in cones without the formation of vesicular structures in wild type and mutant retinae. (a) DAPI overlap of (b). (c) 3D reconstruction of (b). Cone outer segments, as shown by red/green opsin signal (arrow), were attached to the cone inner segments (arrowhead), as shown by GFP signal. (d) DAPI overlap of (e). (f) Single confocal section showing cytoplasmic GFP and membrane bound red/green opsin (see also FIG. 3 ). FIG. 14 further depicts the levels of phosphorylated S6 (p*-S6) (medium gray, light gray marks cones with PNA, dark gray nuclear DAPI stain) in wild type (g, h) and mutant (i-m) cones. (g) Low levels of p*-S6 were seen in wild type cones but not in cone OSs. (h) DAPI overlap of (g). (i, j) Strong uniform expression in cones was seen the PDE-β mutant shortly after the end of the major rod death phase (PW3). Area in lower right corner shows a region where cones had started to die. (j) DAPI overlap of (i). (k-l) Higher magnification at PW5 showing same field at three different confocal depths. (k) Within the plane of the cone outer segments, high levels of p*-S6 were seen in cones when compared to segments of wild type cones. (l) Strong staining was also seen in the plane of the cone nuclei, indicating a uniform cytoplasmic distribution. (m) Within the plane of the INL, levels of p*-S6 were much lower than in cones.
FIG. 15 depicts the affect of insulin levels on cone survival as set forth in Example 1 as follows: (a-c) Retinal flat mounts of PDE-β mutants at PW7 stained for lacZ 49,48 (dark gray) to detect cones (see Material & Methods and FIG. 16 ). (a) Example of untreated control. (b) Example of mouse injected with streptozotocin. (c) Example of mouse injected daily with insulin. (d) Quantification of cone survival after 4 weeks of treatment. Data represents an average of at least 8 retinae and indicates on the y-axis percentage of cone surface area versus surface area of entire retina (see FIGS. 17 and 18 ). (e) Measurements of blood glucose levels and body weight (f) performed weekly over the time span of the experiment. (g, h) Immunofluorescent staining on retinal flat mounts for HIF-1α (medium gray) and PNA (light gray) in untreated control PDE-β −/− (g) and PDE-β −/− mice treated for 4 weeks with insulin (h). Dark gray shows nuclear DAPI.
FIG. 16 depicts the cone-lacZ transgene in the PDE-β mutant at 7 weeks of age as described in Example 1 as follows: (a, b) Double labeling of cones with PNA (dark gray) and lacZ staining medium gray). More cones were labeled by lacZ than by PNA. Since PNA marks an extracellular matrix protein of the OS, once the OSs were reduced, PNA became a less reliable marker. (c, d) Double labeling of cones by α-red/green opsin (dark gray) and lacZ staining (X-gal; medium gray) in the dorsal (c) and ventral (d) retina. Red/green opsin levels decreased ventrally during degeneration which made this marker not suitable for detection of cones across the retina. (e, f) Sections of retina stained for lacZ showing the signal in cones on top of the INL.
FIG. 17 depicts a method to calculate cone survival as described in Example 1 as follows: (a-c) Show retinal flat mounts stained for lacZ (see FIG. 15 ). (a) Untreated control PDE-β−/− mouse at PW7. (b) PDE-β−/− mouse at PW7 treated with one injection of Streptozotocin at PW3. (c) PDE-β−/− mouse at PW7 treated for 4 weeks with daily injections of insulin starting at PW3. (a′-c′) Show inverted color images of corresponding panels (a-c). (a″-c″) Show only the green channels whereas (a′″-c′″) show only the red channels of the inverted color images (a′-c′). The red channel served as a proxy for the lacZ stain whereas the green channel served as a proxy for the retina. (d) Quantification of cone survival by calculating the surface area of red that co-localizes with green. Two different methods were employed, a fixed threshold and an adjusted threshold. The fixed threshold was determined by adjusting the lower intensity of the red channel in the image with the most intense lacZ staining (most intense red channel) to reflect the pattern of the lacZ staining. The same threshold for the red channel was then applied to all other images. As this method would under represent cone survival in mice that were not treated with insulin due to the less intense lacZ staining a second method was employed. For each image the lower intensity of the red channel was adjusted individually to match the blue pattern of the lacZ staining avoiding the problem of the difference in lacZ intensity. The increased intensity of lacZ in the insulin treated mice could be due to healthier cones that either have an increased transcription/translation or decreased protein degradation. (e) Shows the actual calculated values in percentage of cone survival for all retinae. Values are shown for the untreated mice, the Streptozotocin treated mice and the insulin treated mice. Values for both types of calculations are shown, for the fixed threshold and adjusted threshold.
FIG. 18 depicts the assessment of cone survival after prolonged Insulin treatment as described in Example 1 as follows: (a) Composite of retinae after lacZ staining. First column shows untreated PDE-β −/− mice at PW10. Second column shows retinae of PDE-β −/− mice at PW10 that received a single injection of streptozotocin at PW3. Third column shows retinae of PDE-β −/− mice at PW10 that received daily injections of insulin starting at PW3. (b) Shows quantification of cone survival by the two methods described in FIG. 17 . There was no significant difference in cone survival between treated and untreated mice at PW10. (c) Shows comparison between the 4 and 7 weeks treatment.
FIGS. 19A-E depict a table of 230 genes that had statistically significant changes in all 4 mouse models and had fold changes >2 at the onset of cone death, when compared to the other three time points. The fold change is indicated as log 2 . (AVG: Average of fold change from the 4 mutants; C0: Onset of cone death; R: peak of rod death; C1& C2: first and second time point during cone death respectively, see also FIG. 7 a ).
FIG. 20 depicts the injection into the sub-retinal space of the adult eye of an AAV vector containing genes encoding pyruvate carboxylase, fructose 1,6-bisphosphatase and phosphoenolpyruvate carboxykinase in order to induce gluconeogenesis.
FIG. 21 schematically depicts the AAV vector that was used to infect cone cells in the eye of an rd1 mutant mouse.
FIGS. 22A and B depict the mRNA expression and protein expression of gluconeogenesis genes. FIG. 22A shows that Fbp-1, Fbp-2, Pck-1, Pck-2, and Pcx mRNA are expressed in the retina and heart, and that the AAV vector comprising Fbp-1, Pck-1, and Pcx (“construct”; described in Example 2) expresses these genes. Gapdh is used as a loading control and water is used as a negative control. FIG. 22B is a Western blot demonstrating that the protein expression of Fbp-1, Pck-1, and Pcx in HEK 293 cells transfected with the AAV vector comprising these genes is upregulated as compared to the protein expression of these same genes in the retina and HEK 293 control samples.
FIG. 23 is a graph depicting the open field and step tests performed on the rd1 mutant mice in which the AAV vector was introduced.
FIG. 24 is a graph depicting the open field and step tests performed on the rda1 mutant mice in which the AAV vector was introduced.
FIG. 25 depicts Western blot analysis of cellular extracts prepared from retinas transfected with three AAV vectors; one vector comprising Pcx, a second vector comprising Pck-1, and a third vector comprising Fbp-1.
FIG. 26 depicts immunohistochemisty analysis showing overexpression of Pcx in photoreceptors of retinas transfected with three AAV vectors; one vector comprising Pcx, a second vector comprising Pck-1, and a third vector comprising Fbp-1.
FIG. 27 depicts immunohistochemisty analysis showing overexpression of Pcx in photoreceptors of retinas transfected with an AAV vector comprising Pcx and mGFP and a second AAV vector comprising H2BGFP, Fbp-1, and Pck-1.
FIG. 28 depicts immunohistochemisty analysis showing overexpression of Fbp-1 in photoreceptors of retinas transfected with three AAV vectors; one vector comprising Pcx, a second vector comprising Pck-1, and a third vector comprising Fbp-1.
FIG. 29 depicts a map of the AAV2/5 vector comprising the CMV promoter and the gluconeogenesis genes, Pck-1, Fbp-1, and Pcx-1, used to infect cone cells in the eye of an rd1 mutant mouse. See also SEQ ID NO:11.
FIG. 30 depicts a map of the AAV2/5 vector comprising the CAR promoter, the gluconeogenesis gene, Pcx-1, and mGFP. See also SEQ ID NO:12.
FIG. 31 depicts a map of the AAV2/5 vector comprising the CAR promoter and the gluconeogenesis gene, Pck-1. See also SEQ ID NO:13.
FIG. 32 depicts a map of the AAV2/5 vector comprising the CAR promoter, H2BGFP, and the gluconeogenesis genes, Fbp-1 and Pck-1. See also SEQ ID NO:14.
FIG. 33 depicts a map of the AAV2/5 vector comprising the CAR promoter and the gluconeogenesis gene, Fbp-1. See also SEQ ID NO:15.
FIG. 34 depicts a map of the AAV2/5 vector comprising the CAR promoter and the gluconeogenesis gene, Pcx-1. See also SEQ ID NO:16.



DETAILED DESCRIPTION OF THE INVENTION
The present invention is based, at least in part, on the discovery that the upregulation of certain genes in a cell, e.g., a neuronal cell, undergoing cellular starvation, can serve to enhance intracellular levels of glucose, pyruvate, lactate, and/or NADPH. In addition, it has been discovered that cone cells in a mouse model of RP undergo self-digestion, or autophagy, a sign of insufficient nutrition. Accordingly, the upregulation of genes encoding enzymes involved in glucose transport, glucose production, gluconeogenesis, lactate transport and NADPH production can serve to inhibit cellular starvation, thus, increasing cellular viability. Additionally, increased levels of glucose, pyruvate, lactate, and/or NADPH can enhance production of phospholipids, the components of cell membranes, thereby increasing cellular viability under conditions when nutrition is limited.
Without wishing to be bound by any particular theory, it is believed that by upregulating the level of intracellular glucose, pyruvate, lactate, and/or NADPH, the cells are provided with additional nutrition to sustain themselves and, are further provided the building blocks for cellular structure, for example, the building blocks for the production of phospholipids, the primary component of cellular membranes. Accordingly, the enhancement of intracellular glucose, pyruvate, lactate, and/or NADPH serves to promote or enhance cellular viability, e.g., when cells are compromised by genetic and environmental factors and to treat or prevent a disorder associated with starvation of cells, e.g., a disorder that would otherwise lead to malfunction and death of the cells.
Accordingly, the present invention provides methods for inhibiting starvation of a cell, as well as methods for the treatment and/or prevention of disorders associated with cellular starvation, for example, retinitis pigmentosa, by enhancing the intracellular levels of glucose, pyruvate, lactate and/or NADPH.
In one embodiment of the invention, cells suitable for use in the instant methods are neuronal cells. As used herein, the terms “neuron” or “neuronal cell” refer to a nerve cell capable of receiving and conducting electrical impulses from the nervous system. A nerve cell or “neuron” typically comprises a cell body, an axon, axon terminals, and dendrites and is readily identifiable by one of ordinary skill in the art.
As used herein, the terms ″neural″ or “neural cell” also include “glial cells”, also referred to as “neuroglia” or “glia”, which are cells that provide support and nutrition (e.g., glucose or lactate), maintain homeostasis, form myelin, and participate in signal transmission in the nervous system.
The types of glial cells are: “astrocytes”, “oligodendrocytes”, “Schwann cells”), and “microglia”. “Astrocytes” have numerous projections that anchor neurons to their blood supply and regulate nutrition of neuronal cells. They also regulate the external chemical environment of neurons by removing excess ions, notably potassium, and recycling neurotransmitters released during synaptic transmission. “Oligodendrocytes” and “Schwann cells” coat axons in the central or peripheral nervous system, respectively, to form a myelin sheath which provides insulation to the axon that allows electrical signals to propagate more efficiently. “Microglia” are specialized macrophages capable of phagocytosis.
In one embodiment, a neuron is a “photoreceptor cell”, i.e., a specialized neuron found in the retina. The retina is a thin, transparent tissue containing about 120 million separate rod cells (night vision) and 7 million cone cells (day and color vision) as well as millions of other structural supporting and interconnecting cells. Photoreceptor cells consist of “rods” and “cones”, which are the photosensitive cells of the retina. The rods contain rhodopsin, the rod photopigment, and the cones contain other distinct photopigments, which respond to light and ultimately trigger a neural discharge in the output cells of the retina, the ganglion cells. Ultimately, this signal is registered as a visual stimulus in the visual cortex and other target locations in the brain. The retinal pigment epithelial (RPE) cells produce, store and transport a variety of factors that are responsible for the normal function and survival of photoreceptors. Retinal neurons that can also sense light consist of photosensitive ganglion cells. These cells, known as the melanopsin ganglion cells are found in the inner retina, have dendrites and long axons projecting to the protectum (midbrain), the suprachiasmatic nucleas in the hypothalamus, and the lateral geniculate (thalamus). In one embodiment, a photoreceptor cell is a rod. In one embodiment, a photoreceptor cell is a cone. In one embodiment, a photosensitive cell is a cell is a melanopsin ganglion cell.
As used herein, the term “starvation of a cell”, “cellular starvation”, “cellular ischemia” refers to the insufficient supply of nutrients to a cell to allow for proper functioning and maintenance. In particular embodiments, cellular starvation includes insufficient supply of glucose, pyruvate, lactate, NADPH, and/or oxygen and/or insufficient ability to generate phospholipids, cellular membranes, cellular proteins, nucleic acid, carbohydrates, vitamins, or intermediates thereof, and slower than normal cellular processes, e.g., DNA replication, translation, generation of glucose, pyruvate, lactate, NADPH, oxygen, phospholipids, cellular membranes, cellular proteins, nucleic acid, carbohydrates, vitamins, or intermediates thereof. Methods for identifying a cell undergoing starvation are routine to one of ordinary skill in the art and include, for example, determination of the rate of cell division, protein synthesis, glucose uptake, intracellular oxygen levels, organelle digestion (macroautophagy), and protein degradation through, e.g., chaperone-mediated autophagy or the ubiquitin-proteasome system.
As used herein, the term “disorders associated with starvation of a cell” includes disorders in which there is insufficient supply of nutrients to the cell to allow for proper functioning and maintenance. Exemplary disorders include disorders, diseases, conditions or injuries in which upregulation of intracellular glucose, pyruvate, lactate, NADPH would be beneficial, e.g., to increase cell viability, such as ischemic disorders, neurodegenerative disorders, ocular disorders, retinal disorders, stroke, heart attack, or wound healing.
As used herein, the term “neurodegenerative disorder” refers to disorders in which neuronal integrity is threatened, for example, where neuronal cells display decreased survival or exhibit an inability or reduced ability to propagate a signal. Neurodegenerative disorders are well known in the art and include, for example, stroke or Alzheimer's Disease.
As used herein, the term “ocular disorder” refers to a disorder of the eye. In a particular embodiment of the invention, the ocular disorder is characterized and/or associated with cellular starvation. For example, ocular disorders include, but are not limited to, retinal disorders, retinitis pigmentosa, age related macular degeneration, cone rod dystrophy, rod cone dystrophy and glaucoma.
As used herein, the term “retinal disorder” refers generally to a disorder of the retina. In one embodiment, the retinal disorder is associated with decreased viability, for example, death, of cone cells, and/or rod cells. Moreover, in a particular embodiment, a retinal disorder is not associated with blood vessel leakage and/or growth, for example, as is the case with diabetic retinopathy, but, instead is characterized primarily by reduced viability of cone cells and/or rod cells. In certain embodiments, the retinal disorder is a genetic disorder. In a particular embodiment, the retinal disorder is retinitis pigmentosa.
As used herein, the term “retinitis pigmentosa” or “RP” is known in the art and encompasses a disparate group of genetic disorders of rods and cones. Retinitis pigmentosa generally refers to retinal degeneration often characterized by the following manifestations: night blindness, progressive loss of peripheral vision, eventually leading to total blindness; ophthalmoscopic changes consist in dark mosaic-like retinal pigmentation, attenuation of the retinal vessels, waxy pallor of the optic disc, and in the advanced forms, macular degeneration. In some cases there can be a lack of pigmentation. Retinitis pigmentosa can be associated to degenerative opacity of the vitreous body, and cataract. Family history is prominent in retinitis pigmentosa; the pattern of inheritance may be autosomal recessive, autosomal dominant, or X-linked; the autosomal recessive form is the most common and can occur sporadically.
As used herein, the terms “Cone-Rod Dystrophy” or “CRD” and “Rod-Cone Dystrophy” or “RCD” refer to art recognized inherited progressive diseases that cause deterioration of the cone and rod photoreceptor cells and often result in blindness. CRD is characterized by reduced viability or death of cone cells followed by reduced viability or death of rod cells. By contrast, RCD is characterized by reduced viability or death of rod cells followed by reduced viability or death of cone cells.
As used herein, the term ″age-related macular degeneration″ also referred to as “macular degeneration” or “AMD”, refers to the art recognized pathological condition which causes blindness amongst elderly individuals. Age related macular degeneration includes both wet and dry forms of ARMD. The dry form of ARMD, which accounts for about 90 percent of all cases, is also known as atrophic, nonexudative, or drusenoid (age-related) macular degeneration. With the dry form of ARMD, drusen typically accumulate in the retinal pigment epithelium (RPE) tissue beneath/within the Bruch's membrane. Vision loss can then occur when drusen interfere with the function of photoreceptors in the macula, which may include reduction of the flow of nutrients from the choroidal vasculature through the RPE to the photoreceptors. The dry form of ARMD results in the gradual loss of vision over many years. The dry form of ARMD can lead to the wet form of ARMD. The wet form of ARMD, also known as exudative or neovascular (age-related) macular degeneration, can progress rapidly and cause severe damage to central vision. The macular dystrophies include Stargardt Disease, also known as Stargardt Macular Dystrophy or Fundus Flavimaculatus, which is the most frequently encountered juvenile onset form of macular dystrophy.
As used herein, the term “glaucoma” has its art recognized meaning, and refers to a group of eye diseases characterized by degeneration of the optic nerve head and visual field loss, often caused by increased intraocular pressure due to blockage of the channel through which aqueous humor drains (chronic or open-angle glaucoma) or by pressure of the iris against the lens (acute or angle-closure glaucoma). The term “glaucoma,” as used herein, includes primary glaucomas, secondary glaucomas, and familial (i.e., inherited glaucomas). The increase in intraocular pressure may result in a reduction of blood flow through the retinal vasculature, thus leading to a reduction in nutrients delivered to retinal neurons.
As used herein, the term ″stroke″ refers to the art recognized pathological condition in which impairment of consciousness and neurological symptom(s) are acutely induced by a cerebrovascular disorder, which includes intracerebral hemorrhages (hypertensive intracerebral hemorrhage and the like), cerebral infarction, transient ischemic attack, subarachnoid hemorrhage, cerebral thrombosis (atherothrombotic cerebral infarction and the like), cerebral embolism (cardiogenic cerebral embolism and the like) and lacunar infarction.
As used herein, the terms “Alzheimer's Disease” or “AD” encompass both non-hereditary and hereditary forms of the disease. Specifically, the terms, as used herein, include the non-hereditary form which is a progressive degenerative disease of the brain primarily associated with aging. The terms further include the hereditary form called familial Alzheimer's disease (FAD). Clinical presentation of AD is characterized by loss of memory, cognition, reasoning, judgment, and orientation. As the disease progresses, motor, sensory, and linguistic abilities are also affected until there is global impairment of multiple cognitive functions. These cognitive losses occur gradually, but typically lead to severe impairment and death in the range of four to twelve years.
The term ″glucose transporter″ as used herein refers to a protein that catalyzes the transport of glucose across a cell membrane. More specifically the glucose transporter facilitates the uptake of glucose into the cytoplasm across the plasma membrane.
The term ″lactate transporter″ as used herein refers to a protein that catalyzes the transport of lactate across a cell membrane. More specifically the lactate transporter facilitates the uptake of lactate into the cytoplasm across the plasma membrane.
As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. A nucleic acid molecule used in the methods of the present invention can be isolated using standard molecular biology techniques. Using all or portion of a nucleic acid sequence of interest as a hybridization probe, nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning. A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
An ″isolated″ nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term ″isolated″ includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an “isolated” nucleic acid molecule is free of sequences which naturally flank the nucleic acid molecule (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid molecule) in the genomic DNA of the organism from which the nucleic acid molecule is derived.
A nucleic acid molecule for use in the methods of the invention can also be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of a nucleic acid molecule of interest. A nucleic acid molecule used in the methods of the invention can be amplified using cDNA, mRNA or, alternatively, genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. Furthermore, oligonucleotides corresponding to nucleotide sequences of interest can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
The nucleic acids for use in the methods of the invention can also be prepared, e.g., by standard recombinant DNA techniques. A nucleic acid of the invention can also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which has been automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071, incorporated by reference herein).
As used herein, an ″antisense″ nucleic acid comprises a nucleotide sequence which is complementary to a ″sense″ nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule, complementary to an mRNA sequence or complementary to the coding strand of a gene. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
In one embodiment, a nucleic acid molecule of the invention is an siRNA molecule. In another embodiment, a nucleic acid molecule of the invention is an shRNA molecule. In one embodiment, a nucleic acid molecule of the invention mediates RNAi.
In another embodiment, a nucleic acid molecule of the invention mediates translational inhibition. RNA interference (RNAi) is a post-transcriptional, targeted gene-silencing technique that uses double-stranded RNA (dsRNA) to degrade messenger RNA (mRNA) containing the same sequence as the dsRNA (Sharp, P. A. and Zamore, P. D. 287. 2431-2432 (2000); Zamore, P. D., et al. Cell 101, 25-33 (2000). Tuschl, T. et al. Genes Dev. 13, 3191-3197 (1999); Cottrell T R, and Doering T L. 2003. Trends Microbiol. 11:37-43; Bushman F. 2003. Mol. Therapy. 7:9-10; McManus M T and Sharp P A. 2002. Nat Rev Genet. 3:737-47). The process occurs when an endogenous ribonuclease cleaves the longer dsRNA into shorter, e.g., 21- or 22-nucleotide-long RNAs, termed small interfering RNAs or siRNAs. The smaller RNA segments then mediate the degradation of the target mRNA. Kits for synthesis of RNAi are commercially available from, e.g. New England Biolabs or Ambion. In one embodiment one or more of the chemistries described herein for use in antisense RNA can be employed in molecules that mediate RNAi.
As used herein, the term ″vector″ refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a ″plasmid″, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes or nucleic acid molecules to which they are operatively linked and are referred to as “expression vectors” or ″recombinant expression vectors″ or simply ″expression vectors″. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. In some embodiments, ″expression vectors″ are used in order to permit pseudotyping of the viral envelope proteins.
Expression vectors are often in the form of plasmids. In the present specification, ″plasmid″ and ″vector″ may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, adeno-associated viruses, lentiviruses), which serve equivalent functions.
As used herein, the term “retrovirus” is used in reference to RNA viruses that utilize reverse transcriptase during their replication cycle. The retroviral genomic RNA is converted into double-stranded DNA by reverse transcriptase. This double-stranded DNA form of the virus is capable of being integrated into the chromosome of the infected cell; once integrated, it is referred to as a ″provirus.″ The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules which encode the structural proteins and enzymes needed to produce new viral particles. At each end of the provirus are structures called ″long terminal repeats″ or ″LTRs.″ LTRs contain numerous regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences needed for replication and integration of the viral genome. LTRs may be several hundred base pairs in length.
The term ″AAV vector″ refers to a vector derived from an adeno-associated virus serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, or AAVX7. ″rAAV vector″ refers to a vector that includes AAV nucleotide sequences as well as heterologous nucleotide sequences. rAAV vectors require only the 145 base terminal repeats in cis to generate virus. All other viral sequences are dispensable and may be supplied in trans (Muzyczka (1992) Curr. Topics Microbiol. Immunol. 158:97). Typically, the rAAV vector genome will only retain the inverted terminal repeat (ITR) sequences so as to maximize the size of the transgene that can be efficiently packaged by the vector. The ITRs need not be the wild-type nucleotide sequences, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, as long as the sequences provide for functional rescue, replication and packaging. In particular embodiments, the AAV vector is an AAV2/5 or AAV2/8 vector. Suitable AAV vectors are described in, for example, U.S. Pat. No. 7,056,502 and Yan et al. (2002) J. Virology 76(5):2043-2053, the entire contents of which are incorporated herein by reference.
As used herein, the term ″lentivirus″ refers to a group (or genus) of retroviruses that give rise to slowly developing disease. Viruses included within this group include HW (human immunodeficiency virus; including but not limited to HW type 1 and HW type 2), the etiologic agent of the human acquired immunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep; the caprine arthritis-encephalitis virus, which causes immune deficiency, arthritis, and encephalopathy in goats; equine infectious anemia virus (EIAV), which causes autoimmune hemolytic anemia, and encephalopathy in horses; feline immunodeficiency virus (FIV), which causes immune deficiency in cats; bovine immune deficiency virus (BIV), which causes lymphadenopathy, lymphocytosis, and possibly central nervous system infection in cattle; and simian immunodeficiency virus (SW), which cause immune deficiency and encephalopathy in sub-human primates. Diseases caused by these viruses are characterized by a long incubation period and protracted course. Usually, the viruses latently infect monocytes and macrophages, from which they spread to other cells. HW, FW, and SW also readily infect T lymphocytes (i.e., T-cells). In one embodiment of the invention, the lentivirus is not HW.
The term ″promoter″ as used herein refers to a recognition site of a DNA strand to which the RNA polymerase binds. The promoter forms an initiation complex with RNA polymerase to initiate and drive transcriptional activity. The complex can be modified by activating sequences termed “enhancers” or inhibitory sequences termed ″silencers″.
The terms ″transformation,″ ″transfection,″ and “transduction” refer to introduction of a nucleic acid, e.g., a viral vector, into a recipient cell.
As used herein, the term ″subject″ includes warm-blooded animals, preferably mammals, including humans. In a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the primate is a human.
As used herein, the various forms of the term ″modulate″ are intended to include stimulation (e.g., increasing or upregulating a particular response or activity) and inhibition (e.g., decreasing or downregulating a particular response or activity).
As used herein, the term 417 contacting″ (i.e., contacting a cell with an agent) is intended to include incubating the agent and the cell together in vitro (e.g., adding the agent to cells in culture) or administering the agent to a subject such that the agent and cells of the subject are contacted in vivo. The term ″contacting″ is not intended to include exposure of cells to an agent that may occur naturally in a subject (i.e., exposure that may occur as a result of a natural physiological process).
As used herein, the term “administering” to a subject includes dispensing, delivering or applying a composition capable of enhancing the intracellular generation and/or uptake of glucose, NADPH, pyruvate, and/or lactate to a subject by any suitable route for delivery of the composition to the desired location in the subject, including delivery by intraocular administration or intravenous administration. Alternatively or in combination, delivery is by the topical, parenteral or oral route, intracerebral injection, intramuscular injection, subcutaneous/intradermal injection, intravenous injection, buccal administration, transdermal delivery and administration by the rectal, colonic, vaginal, intranasal or respiratory tract route.
Various additional aspects of the methods of the invention are described in further detail in the following subsections.
Methods Of The Invention
The present invention provides methods for inhibiting starvation of a cell, e.g., a neuronal cell, which generally comprise contacting a cell with an agent which enhances the intracellular generation and/or uptake of glucose, pyruvate, lactate, and/or NADPH in the cell.
The present invention also provides methods for treating or preventing a disorder associated with starvation of a cell in a subject. The methods generally comprise administering to the subject an agent which enhances the intracellular generation and/or uptake of glucose, pyruvate, lactate, and/or NADPH.
In another aspect, the present invention provides methods for treating or preventing retinitis pigmentosa in a subject. Such methods generally comprise administering to the subject an agent which enhances the intracellular generation and/or uptake of glucose, pyruvate, lactate, and/or NADPH.
The present invention further provides methods for prolonging the viability of a cone cell. The methods generally comprise contacting the cell with an agent which enhances the intracellular generation and/or uptake of glucose, pyruvate, lactate, and/or NADPH.
In one embodiment, the methods described herein can be performed in vitro. For example, intracellular levels of glucose, pyruvate, lactate, and/or NADPH can be modulated in a cell in vitro and then the treated cells can be administered or re-administered to a subject. In one embodiment, the cell is a mammalian cell, e.g., a human cell. For practicing the methods in vitro, cells can be obtained from a subject by standard methods and incubated (e.g., cultured) in vitro with an agent which stimulates intracellular levels of glucose, pyruvate, lactate, and/or NADPH. Methods for isolating cells are well known in the art. The cells can be readministered to the same subject, or another subject which is compatible with the donor of the cells.
For administration of cells to a subject, it may be preferable to first remove residual agents in the culture from the cells before administering them to the subject. This can be done, for example, by gradient centrifugation of the cells or by washing of the tissue. Methods for the ex vivo genetic modification of cells followed by re-administration to a subject are well known in the art and described in, for example, U.S. Pat. No. 5,399,346 the entire contents of which are incorporated herein by reference.
In one embodiment, the invention allows for modulation of intracellular levels of glucose, pyruvate, lactate, and/or NADPH in vivo, by administering to a subject a therapeutically effective amount of an agent as described herein. For example, intracellular levels of glucose, pyruvate, lactate, and/or NADPH can be modulated to treat or prevent a disorder associated with cellular starvation.
The claimed methods of modulation are not meant to include naturally occurring events. For example, the term ″agent″ or ″modulator″ is not meant to embrace endogenous mediators produced by the cells of a subject.
Application of the methods of the invention for the treatment and/or prevention of a disorder can result in curing the disorder, decreasing at least one symptom associated with the disorder, either in the long term or short term or simply a transient beneficial effect to the subject. Accordingly, as used herein, the terms “treat,” “treatment” and “treating” include the application or administration of agents, as described herein, to a subject who is suffering from a disorder associated with starvation of a cell, or who is susceptible to such conditions with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving or affecting such conditions or at least one symptom of such conditions. As used herein, the condition is also “treated” if recurrence of the condition is reduced, slowed, delayed or prevented.
Subjects suitable for treatment using the regimens of the present invention should have or are susceptible to developing disorders associated with cellular starvation, e.g., neuronal cellular starvation, for example, retinal disorders. For example, subjects may be genetically predisposed to development of the disorders. Alternatively, abnormal progression of the following factors including, but not limited to visual acuity, the rate of death of cone and/or rod cells, night vision, peripheral vision, attenuation of the retinal vessels, and other ophthalmoscopic factors associated with retinal disorders such as retinitis pigmentosa may indicate the existence of or a predisposition to a retinal disorders. Other art recognized symptoms or risk factors, as associated with the development of or predisposition to the particular disorder, for example, Alzheimer's Disease or a stroke, heart attack, diabetes, Parkinson's, may be monitored as well known in the art.
The agents, as described herein, may be administered as necessary to achieve the desired effect and depend on a variety of factors including, but not limited to, the severity of the condition, age and history of the subject and the nature of the composition, for example, the identity of the genes or the affected biochemical pathway. In various embodiments, the compositions may be administered at least two, three, four, five or six times a day. Additionally, the therapeutic or preventative regimens may cover a period of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 weeks.
The ability of an agent to upregulate intracellular levels of glucose, pyruvate, lactate, and/or NADPH can be determined as described herein, e.g., by determining the ability of the agent to modulate: cell viability (e.g., modulation of apoptosis), cleavage of LaminA or Caspase 3; expression of Opn1sw, Opn1mw, LAMP-2A, LAMP-2B, or LAMP-2C; protein production of LAMP-2A, LAMP-2B, LAMP-2C, HIF1-α, or GLUT1; phosphorylation of mTOR, S6K1, AMPK, PTEN, or Akt; phospholipid production; production of reactive oxygen species; and/or the expression and protein synthesis of photoreceptor specific opsins.
In various embodiments, the methods of the present invention further comprise monitoring the effectiveness of treatment. For example, visual acuity, the rate of death of cone and/or rod cells, night vision, peripheral vision, attenuation of the retinal vessels, and other ophthalmoscopic changes associated with retinal disorders such as retinitis pigmentosa may be monitored to assess the effectiveness of treatment. Additionally, the rate of death of cells associated with the particular disorder that is the subject of treatment and/or prevention, may be monitored. Alternatively, the viability of such cells may be monitored, for example, as measured by phospholipid production. The assays described in the Examples section below may also be used to monitor the effectiveness of treatment.
In one embodiment, the agent is a nucleic acid molecule. In a particular embodiment, the nucleic acid molecule is a glucose transporter. For example, glucose transporters for use in the present invention may belong to the GLUT family of transporters (including at least one of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, GLUT13, and GLUT14), encoded by the SLC2 family of genes (including at least one of SLC2A1, SLC2A2, SLC2A3, SLC2A4, SLC2A5, SLC2A6, SLC2A7, SLC2A8, SLC2A9, SLC2A10, SLC2A11, SLC2A12, SLC2A13, and SLC2A14), which upregulate the cellular uptake of glucose by facilitated diffusion. In a particular embodiment, the nucleic acid molecule is SLC2A1 encoding the GLUT1 transporter.
The amino acid sequences of the GLUT family of transporters are known and can be found in, for example, GenBank Accession Nos. GI:166795299 (GLUT1; GI:4557851 (GLUT2; GI:5902090 (GLUT3; GI:4507011 (GLUT4); GI:4507013 (GLUT5, isoform 1); GI:207447703 (GLUT5, isoform 2); GI:223029432 (GLUT6, isoform 1); GI:223029430 (GLUT6, isoform 2); GI:134053883 (GLUT7); GI:21361449 (GLUT8); GI:47933387 (GLUT9, isoform 1); GI:47933389 (GLUT9, isoform 2); GI:13540547 (GLUT10); GI:190684655 (GLUT11, isoform a); GI:68226418 (GLUT11, isoform b); GI:68226420 (GLUT11, isoform c); GI:21553331 (GLUT12); GI:203098995 (GLUT13); and GI:23592238 (GLUT14).
The nucleotide sequences of the SLC2 family of transporters are known and can be found in, for example, GenBank Accession Nos. GI:166795298 (SLC2A1); GI:4557850 (SLC2A2); GI:221136810 (SLC2A3); GI:83722278 (SLC2A4); GI:207446701 (SLC2A5, variant 1); GI:207447702 (SLC2A5, variant 2); GI:223029431 (SLC2A6, variant 1); GI:223029429 (SLC2A6, variant 2); GI:134053882 (SLC2A7); GI:51870928 (SLC2A8); GI:47933386 (SLC2A9, variant 1); GI:47933388 (SLC2A9, variant 2); GI:39777591 (SLC2A1); GI:190684654 (SLC2A11, variant 1); GI:190684652 (SLC2A11, variant 2); GI:190684653 (SLC2A11, variant 3); GI:93277101 (SLC2A12); GI:203098994 (SLC2A13); and GI:24475843 (SLC2A14).
In another embodiment, the stimulatory agent is a nucleic acid molecule involved in promoting gluconeogenesis, thereby increasing metabolic flux through gluconeogenesis, and/or reducing metabolic flux through glycolysis. As is well known in the art, gluconeogenesis promotes the generation of glucose, whereas glycolysis promotes the degradation of glucose. Accordingly, the nucleic acid molecule may be a gluconeogenic gene including, but not limited to, pyruvate carboxylase, phosphoenolpyruvate carboxykinase and fructose 1,6-bisphosphatase. In a particular embodiment, one or more gluconeogenic genes may be utilized.
The nucleotide and amino acid sequences of pyruvate carboxylase are known and can be found in, for example, GenBank Accession Nos. GI:106049294 (variant 1), GI:106049291 (variant 2), and GI:106049527 (variant 3). The nucleotide and amino acid sequences of phosphoenolpyruvate carboxykinase are known and can be found in, for example, GenBank Accession Nos. GI:66346720 (variant 1) and GI:66346722 (variant 2). The nucleotide and amino acid sequences of fructose 1,6-bisphosphatase are known and can be found in, for example, GenBank Accession Nos. GI:160298191 (FBP1, variant 1), GI:189083691 (FBP1, variant 2), and GI:22907027 (FBP2).
The present invention further provides methods for increasing intracellular levels of lactate, which, in turn, can serve as an important precursor for gluconeogenesis and the production of glucose. As is well known in the art, lactate is converted to pyruvate, which, in turn, is converted to oxaloacetate for entry into gluconeogenesis and the production of glucose. Accordingly, in one embodiment, the agent may be involved in increasing the uptake of lactate. For example, the agent may be a stimulatory agent, e.g., a nucleic acid molecule that, for example, encodes a lactate transporter (a monocarboxylic acid transporter). In another embodiment, the agent may be an inhibitory agent that downregulates a negative regulator of, for example, a lactate transporter. In yet another embodiment, the agent may be a stimulatory agent, e.g., glutamate, that, for example, increases extracellular lactate concentrations my causing its release from neighboring cells, e.g., Muller glia and/or astrocytes.
The nucleotide and amino acid sequences of members of the monocarboxylic acid transporter family are known and can be found in, for example, GenBank Accession Nos. GI:115583684 (SLC16A1, variant 1) and GI:262073006 (SLC16A1, variant 2); GI:164663748 (SLC16A2); GI:109288011 (SLC16A3, variant 1), GI:109288008 (SLC16A3, variant 2), and GI:109288009 (SLC16A3, variant 3); GI:4759113 (SLC16A4); GI:20127461 (SLC16A5); GI:141802120 (SLC16A6); GI:34222196 (SLC16A7); GI:114796625 (SLC16A8); GI:197383642 (SLC16A9); GI:221139821 (SLC16A10); GI:23503292 (SLC16A11); GI:157041232 (SLC16A12); GI:222537717 (SLC16A13); and GI:42415495 (SLC16A14).
The present invention further provides methods for increasing intracellular levels of NADPH, which, as described above, is important for the generation of phospholipids, the primary component of cellular membranes. Moreover, NADPH serves to detoxify free oxygen radicals which can be damaging to cells, e.g., neuronal cells. For example, after rod cells die, excess oxygen, which is subsequently converted to free oxygen radicals in the presence of light or by phototransduction, accumulates around cone cells. The presence of free oxygen radicals diverts available NADPH from, for example, the generation of phospholipids, thereby reducing the viability of cells. By enhancing the levels of NADPH, in accordance with the methods of the present invention, one can serve to increase phospholipid production and, in turn, cellular membrane generation and, further, to detoxify otherwise damaging free oxygen radicals.
Accordingly, in a particular embodiment, the methods of the present invention are directed to enhancing the ability of a cell to generate NADPH. For example, increasing cellular levels of glucose by the methods described herein serves to increase metabolic flux through the pentose phosphate pathway, thereby generating increased levels of NADPH. Alternatively or in combination, cells suffering from starvation or subject suffering from disorders associated with such starvation can be contacted with agents, e.g., stimulatory agents, which directly enhance intracellular levels of NADPH. For example, the stimulatory agent may be a nucleic acid molecule, e.g., a nucleic acid molecule encoding malic enzyme, which serves to convert malate to pyruvate and generate NADPH as a byproduct. In another embodiment, the stimulatory agent may be, e.g., insulin or triiodothyronine (T3) that e.g., stimulates the expression of malic enzyme. In yet another embodiment, the stimulatory agent may be glutamate that, e.g., stimulates lactate release from neighboring cells.
The nucleotide and amino acid sequences of members of the malic enzyme family are known and can be found in, for example, GenBank Accession Nos. GI:112382261 (ME1), GI:270265877 (ME2, variant 1), GI:270265878 (ME2, variant 2), GI:62420879 (ME3, variant 1), GI:62420881 (ME3, variant 2), and GI:239049446 (ME3, variant 3).
In another embodiment, the agent is an inhibitory agent which downregulates a negative regulator of the synthesis of, for example, malate. In one aspect, the present invention is directed to the use of an agent, e.g., a nucleic acid molecule, vectors and compositions comprising such nucleic acid molecules, to prolong the viability of cone cells. In one embodiment, the viability or survival of cones cells is short term viability, e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 3 years, about 4 years, about 5 years, about 10 years, about 15, years, about 20 years, about 25 years, about 30 years, about 40 years, about 50 years, about 60 years, about 70 years, and about 80 years.
The methods of the invention described above, thus, may be used to treat or prevent starvation of cells and disorders associated with such starvation. In one embodiment, the disorder is a neurodegenerative disorder, such as a stroke or Alzheimer's Disease. In other embodiments, the disorder is an ocular disorder including, but not limited to, retinitis pigmentosa, age related macular degeneration, cone rod dystrophy, rod cone dystrophy and glaucoma. In further embodiments, the disorder is an ocular disorder associated with decreased viability of cone and/or rod cells. In yet another embodiment, the disorder is a genetic disorder.
In one embodiment, the invention is directed to a method of treating or preventing a disorder associated with starvation of a neuronal cell, for example, retinitis pigmentosa, in a subject by selecting a subject who is susceptible to the development of the disorder and administering to the subject an effective amount of the nucleic acid molecules, vectors and/or compositions of the present invention, thereby treating or preventing the disorder in the subject.
The overall strategy to save neurons from degeneration is to supply them with the genes that will allow them to make up for deficits in the building materials that are required to maintain function and survival. Genes encoding gluconeogenic enzymes, glucose transporter(s), and/or NADPH synthetic enzymes are those targeted for delivery. Vectors derived from AAV, adenoviruses, lentiviruses and/or other types of retroviruses, as well as electroporation can be used.
Age related macular degeneration is another disease in which cones die. The early signs of this disease are drusen, which are accumulations of lipids and proteins in the region between the choroidal vasculature, the retinal pigmented epithelium, and photoreceptors. The drusen likely impedes the flow of nutrients through the RPE to the photoreceptors. Treatments with the gluoconeogenic enzymes would prevent the rapid death of nutritionally deprived cones. In addition to cone photoreceptor survival, the survival of several other types of neurons has been proposed to result form lack of glucose and/or oxygen, or other nutrients.
Glaucoma is another disease that can be treated using the methods of the present invention. Glaucoma is a disease in which ganglion cells die, and high intraocular pressure often accompanies ganglion cell death. Compromised blood flow due to increased pressure might cause the ganglion cells to die. Gluconeogenic enzymes are expected to prolong their survival by allowing lactate made by Muller glial cells to be utilized for glucose synthesis.
Stroke is yet another disease that can be treated using the methods of the present invention. Stroke is caused by a compromised blood supply. Given that neurons expend energy rapidly, a depletion in glucose and oxygen can lead to rapid death. Supplying the genes for gluconeogenesis quickly after a stroke will prevent neuronal death.
Similarly, heart attack is caused, at least in part, by compromised blood supply and, thus, depletion in glucose and oxygen often leading to heart muscle death. Accordingly, supplying the genes for gluconeogenesis quickly after a heart attack will prevent heart cell death
Alzheimer's Disease may be caused, at least in part, by a reduction in energy supply. Physical and mental activity later in life is believed to supply good blood flow to the areas being used, thereby helping those cells maintain sufficient nutrition to prevent neuronal death. Providing cells with gluconeogenic genes is expected to also prevent this type of degeneration.
Injuries, such as burns and other wounds will also be benefited by the methods of the invention in that increases in glucose, pyruvate, lactate, and/or NADPH will allow the production of all cellular intermediates for cellular repair necessary for wound healing.
Agents for Use in the Methods of the Invention
Stimulatory Agents
The methods of the invention may use stimulatory agents which upregulate or enhance the intracellular generation and/or uptake of glucose, pyruvate, lactate, and/or NADPH in a cell. Examples of such stimulatory agents include proteins, nucleic acid molecules, e.g., expression vectors comprising nucleic acid molecules, and chemical agents that stimulate expression and/or activity of a protein which enhances the intracellular generation and/or uptake of glucose, pyruvate, lactate, and/or NADPH in a cell.
A preferred stimulatory agent is a nucleic acid molecule encoding a protein of interest. For example, a cDNA (full length or partial cDNA sequence) is cloned into a recombinant expression vector and the vector is transfected into cells using standard molecular biology techniques. The cDNA can be obtained, for example, by amplification using the polymerase chain reaction (PCR) or by screening an appropriate cDNA library.
Following isolation or amplification of a cDNA, the DNA fragment is introduced into a suitable expression vector. For example, nucleic acid molecules encoding a protein of interest in the form suitable for expression of the protein in a host cell, can be prepared using nucleotide sequences based on the nucleic acid sequence of a nucleic acid molecule encoding the protein of interest.
In one embodiment, a stimulatory agent can be present in an inducible construct. In another embodiment, a stimulatory agent can be present in a construct which leads to constitutive expression.
In one embodiment, the nucleic acid molecules of the invention may be delivered to cells, e.g., neuronal cells, or to subjects using a viral vector, preferably one whose use for gene therapy is well known in the art. Techniques for the formation of vectors or virions are generally described in ″Working Toward Human Gene Therapy,″ Chapter 28 in Recombinant DNA, 2nd Ed., Watson, J. D. et al., eds., New York: Scientific American Books, pp. 567-581 (1992). An overview of suitable viral vectors or virions is provided in Wilson, J. M., Clin. Exp. Immunol. 107(Suppl. 1):31-32 (1997), as well as Nakanishi, M., Crit. Rev. Therapeu. Drug Carrier Systems 12:263-310 (1995); Robbins, P. D., et al., Trends Biotechnol. 16:35-40 (1998); Zhang, J., et al., Cancer Metastasis Rev. 15:385-401 (1996); and Kramm, C. M., et al., Brain Pathology 5:345-381 (1995). Such vectors may be derived from viruses that contain RNA (Vile, R. G., et al., Br. Med. Bull. 51:12-30 (1995)) or DNA (Ali M., et al., Gene Ther. 1:367-384 (1994)).
Examples of viral vector systems utilized in the gene therapy art and, thus, suitable for use in the present invention, include the following: retroviruses (Vile, R. G., supra; U.S. Pat. Nos. 5,741,486 and 5,763,242); adenoviruses (Brody, S. L., et al., Ann. N.Y. Acad. Sci. 716: 90-101 (1994); Heise, C. et al., Nat. Med. 3:639-645 (1997)); adenoviral/retroviral chimeras (Bilbao, G., et al., FASEB J. 11:624-634 (1997); Feng, M., et al., Nat. Biotechnol. 15:866-870 (1997)); adeno-associated viruses (Flotte, T. R. and Carter, B. J., Gene Ther. 2:357-362 (1995); U.S. Pat. No. 5,756,283); herpes simplex virus I or II (Latchman, D. S., Mol. Biotechnol. 2:179-195 (1994); U.S. Pat. No. 5,763,217; Chase, M., et al., Nature Biotechnol. 16:444-448 (1998)); parvovirus (Shaughnessy, E., et al., Semin Oncol. 23:159-171 (1996)); reticuloendotheliosis virus (Donburg, R., Gene Therap. 2:301-310 (1995)). Extrachromosomal replicating vectors may also be used in the gene therapy methods of the present invention. Such vectors are described in, for example, Calos, M. P. (1996) Trends Genet. 12:463-466, the entire contents of which are incorporated herein by reference. Other viruses that can be used as vectors for gene delivery include poliovirus, papillomavirus, vaccinia virus, lentivirus, as well as hybrid or chimeric vectors incorporating favorable aspects of two or more viruses (Nakanishi, M. (1995) Crit. Rev. Therapeu. Drug Carrier Systems 12:263-310; Zhang, J., et al. (1996) Cancer Metastasis Rev. 15:385-401; Jacoby, D. R., et al. (1997) Gene Therapy 4:1281-1283).
In a particular embodiment, the viral vector for use in the methods of the present invention is an AAV vector. In particular embodiments, the viral vector is an AAV2/5 or AAV2/8 vector. Such vectors are described in, for example, U.S. Pat. No. 7,056,502, the entire contents of which are incorporated herein by reference.
The vector will include one or more promoters or enhancers, the selection of which will be known to those skilled in the art. Suitable promoters include, but are not limited to, the retroviral long terminal repeat (LTR), the SV40 promoter, the human cytomegalovirus (CMV) promoter, and other viral and eukaryotic cellular promoters known to the skilled artisan.
Guidance in the construction of gene therapy vectors and the introduction thereof into affected animals for therapeutic purposes may be obtained in the above-referenced publications, as well as in U.S. Pat. Nos. 5,631,236, 5,688,773, 5,691,177, 5,670,488, 5,529,774, 5,601,818, and PCT Publication No. WO 95/06486, the entire contents of which are incorporated herein by reference.
Generally, methods are known in the art for viral infection of the cells of interest. The virus can be placed in contact with the neuronal cell of interest or alternatively, can be injected into a subject suffering from a disorder associated with neuronal cellular starvation.
In one aspect of the invention, the therapeutic nucleic acid molecule or the vector containing the same will be in the form of a pharmaceutical composition containing a pharmaceutically acceptable carrier. As used herein ″pharmaceutically acceptable carrier″ includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for intraocular, parenteral, intravenous, intraperitoneal, topical, or intramuscular administration. In another embodiment, the carrier is suitable for oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the gene therapy vector, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
In a particular embodiment, the pharmaceutical compositions of the present invention would be administered in the form of injectable compositions. The vector can be prepared as an injectable, either as liquid solutions or suspensions. The preparation may also be emulsified. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the preparation may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH-buffering agents, adjuvants or immunopotentiators.
In a particular embodiment, the nucleic acid molecules and/or vectors are incorporated in a composition suitable for intraocular administration. For example, the compositions may be designed for intravitreal, subconjuctival, sub-tenon, periocular, retrobulbar, suprachoroidal, and/or intrascleral administration, for example, by injection, to effectively treat the retinal disorder. Additionally, a sutured or refillable dome can be placed over the administration site to prevent or to reduce ″wash out″, leaching and/or diffusion of the active agent in a non-preferred direction.
Relatively high viscosity compositions, as described herein, may be used to provide effective, and preferably substantially long-lasting delivery of the nucleic acid molecules and/or vectors, for example, by injection to the posterior segment of the eye. A viscosity inducing agent can serve to maintain the nucleic acid molecules and/or vectors in a desirable suspension form, thereby preventing deposition of the composition in the bottom surface of the eye. Such compositions can be prepared as described in U.S. Pat. No. 5,292,724, the entire contents of which are hereby incorporated herein by reference.
In general, the nucleic acid molecule is provided in a therapeutically effective amount to elicit the desired effect, e.g., enhancing intracellular levels of glucose, pyruvate, lactate, and/or NADPH. The quantity of the vector to be administered, both according to number of treatments and amount, will also depend on factors such as the clinical status, age, and weight of the subject to be treated, and the severity of the disorder. Precise amounts of active ingredient required to be administered depend on the judgment of the gene therapist and will be particular to each individual patient. Generally, the viral vector is administered in titers ranging from about 1×10 5 , about 1.5×10 5 , about 2×10 5 , about 2.5×10 5 , about 3×10 5 , about 3.5×10 5 , about 4×10 5 , about 4.5×10 5 , about 5×10 5 , about 5.5×10 5 , about 6×10 5 , about 6.5×10 5 , about 7×10 5 , about 7.5×10 5 , about 8×10 5 , about 8.5×10 5 , about 9×10 5 , about 9.5×10 5 , about 1×10 6 , about 1.5×10 6 , about 2×10 6 , about 2.5×10 6 , about 3×10 6 , about 3.5×10 6 , about 4×10 6 , about 4.5×10 6 , about 5×10 6 , about 5.5×10 6 , about 6×10 6 , about 6.5×10 6 , about 7×10 6 , about 7.5×10 6 , about 8×10 6 , about 8.5×10, about 9×10 6 , about 9.5×10 6 , about 1×10 7 , about 1.5×10 7 , about 2×10 7 , about 2.5×10 7 , about 3×10 7 , about 3.5×10 7 , about 4×10 7 , about 4.5×10 7 , about 5×10 7 , about 5.5×10 7 , about 6×10 7 , about 6.5×10 7 , about 7×10 7 , about 7.5×10 7 , about 8×10 7 , about 8.5×10 7 , about 9×10 7 , about 9.5×10 7 , about 1×10 8 , about 1.5×10 8 , about 2×10 8 , about 2.5×10 8 , about 3×10 8 , about 3.5×10 8 , about 4×10 8 , about 4.5×10 8 , about 5×10 8 , about 5.5×10 8 , about 6×10 8 , about 6.5×10 8 , about 7×10 8 , about 7.5×10 8 , about 8×10 8 , about 8.5×10 8 , about 9×10 8 , about 9.5×10 8 , and about 1×10 9 colony forming units (cfu) per ml, although ranges may vary. Preferred titers will range from about 1×10 6 to about 1×10 8 cfu/ml.
In one embodiment, a packaging cell line is transduced with a retroviral vector carrying the desired nucleic acid molecule to form a producer cell line. The packaging cells may be transduced by any means known in the art, including, e.g., electroporation, CaPO 4 precipitation, or the use of liposomes. Examples of packaging cells that may be transfected include, but are not limited to, BOSC23, Bing, PE501, PA317, .PSI.-2, .PSI.-AM, PA12, T19-14X, VT-19-17-H2, .PSI.-CRE, .PSI.-CRIP, GP+E86, GP+envAm12, and DAN cell lines. Guidance on retroviral producing packaging cells and how to construct them can be found in Short et al., J. Neurosci. Res. 27:427-433 (1990); Miller, A. D., Human Gene Ther. 1:5-14 (1990); Danos, 0, ″Construction of Retroviral Packaging Cell Lines,″ in Methods in Molecular Biology (M. Collins, ed.), Vol. 8, The Humana Press Inc., Clifton, N.J., 17-26 (1991); Murdoch, B., et al., Gene Therapy 4:744-749 (1997); and U.S. Pat. Nos. 5,529,774 and 5,591,624, the entire contents of which are incorporated herein by reference.
Retroviral vectors have also been successfully packaged with a vesicular stomatitis virus (VSV) envelope glycoprotein G (″pseudotyping″). These vectors are more stable and can be concentrated to 10 9 cfu/ml, allowing them to be injected directly (Burns, J. C. et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037).
The producer cells can then be grafted near or into the desired location, for example, intraocularly. Direct injection of high titer retroviral producer cells (Murdoch, B., et al., Gene Ther. 4:744-749 (1997); Onodera, M., et al., Hum Gene Ther. 8:1189-1194 (1997)) should allow for efficient in situ infection with the retroviral sequences (Rainov, N. G., et al., Cancer Gene Ther. 3:99-106 (1996); Ram, Z., et al., Cancer Res. 53:83-88 (1993)). Producer cells injected intraocularly do not generally migrate from the site of injection. Moreover, although they may be rejected by the host, this does not occur for 5-10 days, by which time retroviral infection of nearby cells will have occurred (Ram, Z., et al., J. Neurosurg. 79:400-407 (1993)). In general, vector producer cell (VPC) dosages range from about 2.5×10 8 , about 1×10 8 , about 1.5×10 8 , about 2×10 8 , about 2.5×10 8 , about 3×10 8 , about 3.5×10 8 , about 4×10 8 , about 4.5×10 8 , about 5×10 8 , about 5.5×10 8 , about 6×10 8 , about 6.5×10 8 , about 7×10 8 , about 7.5×10 8 , about 8×10 8 , about 8.5×10 8 , about 9×10 8 , about 9.5×10 8 , and about 1×10 9 VPCs. The exact amount of producer cells will ultimately be determined by the skilled artisan based on numerous factors, including, but not limited to, the available injectable volume, clinical status of the patient, and the severity of the disorder.
Preferably, the viral genomes of the viral vectors used in the invention should be modified to remove or limit their ability to replicate, however, replication conditional viruses will also be useful in the present invention, as will replicating vectors that are capable of targeting certain cells. (See, e.g., Zhang, J. et al. (1996) Cancer Metastasis Rev. 15:385-401).
In one embodiment, a single viral vector is used to carry multiple nucleic acid molecules, for example, genes encoding pyruvate carboxylase and phosphoenolpyruvate carboxykinase. In another embodiment, two viral vectors are used each carrying one or more genes of interest. If two viral vectors are used, they can be derived from the same or a different type of virus, and can be administered simultaneously or sequentially (i.e., without regard for a specific order).
The nucleic acid molecules can also be delivered using non-viral methods for gene transfer, preferably those whose use in gene therapy is known in the art (Nakanishi, M., Crit. Rev. Therapeu. Drug Carrier Systems 12:263-310 (1995); Abdallah, B., et al., Biol Cell 85:1-7 (1995); Zhang, J., et al., Cancer Metastasis Rev. 15:385-401 (1996); Philips, S. C., Biologicals 23:13-16 (1995); Lee, R. J. and Huang, L., Crit. Rev. Ther. Drug Carrier Syst. 14:173-206 (1997)). Examples of such non-viral vectors for gene delivery include prokaryotic vectors, cationic liposomes, DNA-protein complexes, non-viral T7 autogene vectors (Chen, X., et al., Hum. Gene Ther. 9:729-736 (1998)), fusogenic liposomes, direct injection of nucleic acid (″naked DNA″), particle or receptor-mediated gene transfer, hybrid vectors such as DNA-adenovirus conjugates or other molecular conjugates involving a non-viral and viral component, starburstpolyamidoamine dendrimers (Kukowska-Latallo, J. F., et al., Proc Natl Acad Sci USA 93:4897-4902 (1996); Tang, M. X., et al., Bioconjug. Chem. 7:703-714 (1996)), cationic peptides (Wyman, T. B., et al., Biochemistry 36:3008-3017 (1997)), mammalian artificial chromosomes (Ascenzioni, F., et al., Cancer Lett. 118:135-142 (1997)), and nanoparticles (Parker Read et al. J. Gene Med. 12:86-96 (2010); Frajo et al. PlosOne 1:E38 (2006).
In addition, the present invention provides an embodiment of the foregoing methods wherein the nucleic acid molecules are delivered using any cellular vector, preferably one whose use for gene therapy is well-established for those skilled in the art. Examples of such cellular vectors for gene therapy include endothelial cells (Rancourt, C., et al., Clin. Cancer Res. 4:265-270 (1998); Qjeifo, J. O., et al., Cytokines Mol. Ther. 2:89-101 (1996)) and macrophages including tumor-infiltrating macrophages (Zufferey, R., et al., Nat. Biotechnol. 15:871-875 (1997); Naldini, L., et al., Science 272:263-267 (1996)), each of which may be modified using viral or non-viral vectors to carry the desired nucleic acid molecules, and thus express the desired gene products. Other suitable non-viral vectors will be readily apparent to the skilled artisan.
Gene delivery can be enhanced by including an internal ribosome entry site (IRES) sequence to achieve coordinate expression of multiple genes on a bicistronic message. IRESs are sequences containing 500-600 bp that are typical of the 5′ nontransduced regions of picornaviruses, including the polio- and encephalomyocarditis viruses (EMCV). See, e.g., Ghattas, I. R., et al., Molecular and Cellular Biology 11:5848-5859 (1991); Morgan, R. A., et al., Nucleic Acids Research 20:1293-1299 (1992). This approach has been used for efficient retroviral coexpression of the two subunits of interleukin-12 (Tahara, H., et al., J. Immunol. 154:6466-6474 (1995)). Similarly, a viral sequence, the picornavirus 2A sequence, can be used to create mRNAs encoding more than one protein. The viral 2A peptide is 16-20 amino acids and can be employed as a cleavage peptide located between two proteins of interest, where it promotes their cleavage into two separate proteins (Furler et al. Gene Ther. 8:864-873 (2001). Another alternative is for the vector to contain multiple genes under the control of distinct promoters.
Other examples of stimulatory agents for enhancing the intracellular generation and/or uptake of glucose, pyruvate, lactate, and/or NADPH in a cell is a small molecule compound, an antibody, or other protein as described below.
Inhibitory Agents
The methods of the invention may also use agents which inhibit a negative regulator of the intracellular generation and/or uptake of glucose, pyruvate, lactate, and/or NADPH. Such agents can be, for example, intracellular binding molecules that act to specifically inhibit the expression, processing, post-translational modification, or activity of a negative regulator of the intracellular generation and/or uptake of glucose, pyruvate, lactate, and/or NADPH. As used herein, the term ″intracellular binding molecule″ is intended to include molecules that act intracellularly to, for example, inhibit the processing expression or activity of a protein by binding to the protein or to a nucleic acid (e.g. an mRNA molecule) that encodes the protein.
Examples of intracellular binding molecules, described in further detail below, include antisense nucleic acids, intracellular antibodies, peptidic compounds, and chemical agents that specifically inhibit the activity of a negative regulator of intracellular levels of glucose, pyruvate, lactate, and/or NADPH.
In one embodiment, such an agent is an antisense nucleic acid molecule that is complementary to a gene encoding a negative regulator of intracellular levels of glucose, pyruvate, lactate, and/or NADPH, or to a portion of said gene, or a recombinant expression vector encoding the antisense nucleic acid molecule. The use of antisense nucleic acids to downregulate the expression of a particular protein in a cell is well known in the art (see e.g. Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews—Trends in Genetics, Vol. 1(1) 1986; Askari, F. K. and McDonnell, W. M. (1996) N. Eng J. Med. 334:316-318; Bennett, M. R. and Schwartz, S. M. (1995) Circulation 92:1981-1993; Mercola, D. and Cohen, J. S. (1995) Cancer Gene Ther._:47-59; Rossi, J. J. (1995) Br. Med. Bull. 51:217-225; Wagner, R. W. (1994) Nature 372:333-335; each of which is incorporated herein by reference). An antisense nucleic acid molecule comprises a nucleotide sequence that is complementary to the coding strand of another nucleic acid molecule (e.g., an mRNA sequence) and accordingly is capable of hydrogen bonding to the coding strand of the other nucleic acid molecule.
Antisense sequences complementary to a sequence of an mRNA can be complementary to a sequence found in the coding region of the mRNA, the 5′ or 3′ untranslated region of the mRNA or a region bridging the coding region and an untranslated region (e.g. at the junction of the 5′ untranslated region and the coding region). Furthermore, an antisense nucleic acid can be complementary in sequence to a regulatory region of the gene encoding the mRNA, for instance a transcription initiation sequence or regulatory element. Preferably, an antisense nucleic acid is designed so as to be complementary to a region preceding or spanning the initiation codon on the coding strand or in the 3′ untranslated region of an mRNA.
Antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of an mRNA, but more preferably is antisense to only a portion of the coding or noncoding region of an mRNA.
An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g. an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g. phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. To inhibit expression in cells, one or more antisense oligonucleotides can be used.
Alternatively, an antisense nucleic acid can be produced biologically using an expression vector into which all or a portion of a cDNA has been subcloned in an antisense orientation (i.e., nucleic acid transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest). Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the expression of the antisense RNA molecule in a cell of interest, for instance promoters and/or enhancers or other regulatory sequences can be chosen which direct constitutive, tissue specific or inducible expression of antisense RNA. The antisense expression vector is prepared according to standard recombinant DNA methods for constructing recombinant expression vectors, except that the cDNA (or portion thereof) is cloned into the vector in the antisense orientation. The antisense expression vector can be in the form of, for example, a recombinant plasmid, phagemid or attenuated virus. The antisense expression vector can be introduced into cells using a standard transfection technique.
Antisense nucleic acid molecules are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a protein to thereby inhibit expression of the protein, e.g. by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarily to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of an antisense nucleic acid molecule of the invention includes direct injection at a tissue site. Alternatively, an antisense nucleic acid molecule can be modified to target selected cells and then administered systemically. For example, for systemic administration, an antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, e.g. by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen. The antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
In yet another embodiment, an antisense nucleic acid molecule that may be used in the methods of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual, 8-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641; incorporated herein by reference). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148; incorporated herein by reference) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330; incorporated herein by reference).
In still another embodiment, an antisense nucleic acid molecule that may be used in the methods of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g. hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591; incorporated herein by reference)) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation mRNAs. A ribozyme having specificity for an encoding nucleic acid molecule of interest can be designed based upon the nucleotide sequence of the cDNA. For example, a derivative of a Tetrahynena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in, an encoding mRNA of interest. See, e.g. Cech et al. U.S. Pat. No. 4,987,071; Cech et al. U.S. Pat. No. 5,116,742; each of which is incorporated herein by reference. Alternatively, a mRNA of interest can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418; incorporated herein by reference.
In another embodiment, a agent that promotes RNAi can be used to inhibit expression of a negative regulator of intracellular levels of glucose, pyruvate, lactate, and/or NADPH. RNA interference (RNAi is a post-transcriptional, targeted gene-silencing technique that uses double-stranded RNA (dsRNA) to degrade messenger RNA (mRNA) containing the same sequence as the dsRNA (Sharp, P. A. and Zamore, P. D. 287. 2431-2432 (2000); Zamore et al. Cell 101, 25-33 (2000). Tuschl et al. Genes Dev. 13. 3191-3197 (1999); Cottrell T R, and Doering T L. 2003. Trends Microbiol. 11:37-43; Bushman F. 2003. Mol. Therapy. 7:9-10; McManus M T and Sharp P A. 2002. Nat. Rev. Genet. 3:737-47; each of which is incorporated herein by reference). The process occurs when an endogenous ribonuclease cleaves the longer dsRNA into shorter, e.g. 21- or 22-nucleotide-long RNAs, termed small interfering RNAs or siRNAs. The smaller RNA segments then mediate the degradation of the target mRNA. Kits for synthesis of RNAi are commercially available from, e.g. New England Biolabsor Ambion. In one embodiment one or more of the chemistries described above for use in antisense RNA can be employed in molecules that mediate RNAi.
Antibodies can also be used as agents in the methods of the invention. In one embodiment, an antibody is an intracellular antibody that inhibits protein activity. Such an intracellular antibody is prepared using methods well known in the art which generally involve preparing a recombinant expression vector which encodes the antibody chains in a form such that, upon introduction of the vector into a cell, the antibody chains are expressed as a functional antibody in an intracellular compartment of the cell.
For inhibition of transcription factor activity according to the inhibitory methods of the invention, an intracellular antibody that specifically binds the protein is expressed within the nucleus of the cell. Nuclear expression of an intracellular antibody can be accomplished by removing from the antibody light and heavy chain genes those nucleotide sequences that encode the N-terminal hydrophobic leader sequences and adding nucleotide sequences encoding a nuclear localization signal at either the N- or C-terminus of the light and heavy chain genes (see e.g. Biocca et al. (1990) EMBO J. 9:101-108; Mhashilkar et al. (1995) EMBO J. 14:1542-1551; each of which is incorporated herein by reference). A preferred nuclear localization signal to be used for nuclear targeting of the intracellular antibody chains is the nuclear localization signal of SV40 Large T antigen (see Biocca et al. (1990) EMBO J. 9:101-108; Mhashilkar et al. (1995) EMBO J. 14:1542-1551; each of which is incorporated herein by reference).
To prepare an intracellular antibody expression vector, antibody light and heavy chain cDNAs encoding antibody chains specific for the target protein of interest, is isolated, typically from a hybridoma that secretes a monoclonal antibody specific for the protein. Antibodies can be prepared by immunizing a suitable subject, (e.g. rabbit, goat, mouse or other mammal), e.g., with a protein immunogen. An appropriate immunogenic preparation can contain, for example, recombinantly expressed protein or a chemically synthesized peptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory compound. Antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497; incorporated herein by reference) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J Biol Chem 255:4980-83; Yeh et al. (1976) PNAS76:2927-31; Yeh et al. (1982) Int. J. Cancer 29:269-75; each of which is incorporated herein by reference). The technology for producing monoclonal antibody hybridomas is well known (see generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet., 3:231-36; each of which is incorporated herein by reference). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a protein immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds specifcally, a protein of interest. Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating a monoclonal antibody (see, e.g. G. Galfre et al. (1977) Nature 266:550-52; Gefter et al. Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited supra; each of which is incorporated herein by reference). Moreover, the ordinary skilled artisan will appreciate that there are many variations of such methods which also would be useful.
Typically, the immortal cell line (e.g. a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine (″HAT medium″). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g. the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O— Ag14 myeloma lines. These myeloma lines are available from the American Type Culture Collection (ATCC), Rockville, Md. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (″PEG″). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody that specifically binds the protein are identified by screening the hybridoma culture supernatants for such antibodies, e.g. using a standard ELISA assay.
Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody that binds to a protein can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g. an antibody phage display library) with the protein, or a peptide thereof, to thereby isolate immunoglobulin library members that bind specifically to the protein. Kits for generating and screening phage display libraries are commercially available (e.g. the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurJZAP™ Phage Display Kit, Catalog No. 240612; each of which is incorporated herein by reference).
Examples of methods and compounds particularly amenable for use in generating and screening antibody display libraries can also be found in, for example, Ladner et al U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Grifeths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) NucAcid Res 19:4133-4137; Barbas et al. (1991) PNAS 88:7978-7982; McCafferty et al. Nature (1990) 348:552-554; each of which is incorporated herein by reference.
In another embodiment, ribosomal display can be used to replace bacteriophage as the display platform fro identifying antibodies for use in the methods of the invention (see, e.g. Hanes et al. 2000. Nat. Biotechnol. 18:1287; Wilson et al. 2001. Proc. Natl. Acad. Sci. USA 98:3750; Irving et al. 2001 J. Immunol. Methods 248:31; each of which is incorporated herein by reference). In yet another embodiment, cell surface libraries can be screened for antibodies (Boder et al. 2000. Proc. Natl. Acad. Sci. USA 97: 10701; Daugherty et al. 2000 J. Immunol. Methods 243:211; each of which is incorporated herein by reference). Such procedures provide alternatives to traditional hybridoma techniques for the isolation and subsequent cloning of monoclonal antibodies.
In another embodiment, an antibody that may be used in the methods of the invention is a substantially human antibody generated in transgenic animals (e.g., mice) that are incapable of endogenous immunoglobulin production (see e.g. U.S. Pat. Nos. 6,075,181, 5,939,598, 5,591,669 and 5,589,369 each of which is incorporated herein by reference).
For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of a human immunoglobulin gene array to such germ line mutant mice will result in the production of human antibodies upon antigen challenge. Another preferred means of generating human antibodies using SCID mice is disclosed in U.S. Pat. No. 5,811,524 which is incorporated herein by reference. It will be appreciated that the genetic material associated with these human antibodies can also be isolated and manipulated as described herein.
Yet another highly efficient means for generating recombinant antibodies for use in the methods of the invention is disclosed by Newman, Biotechnology, 10:1455-1460 (1992); incorporated herein by reference. Specifically, this technique results in the generation of primatized antibodies that contain monkey variable domains and human constant sequences. This reference is incorporated by reference in its entirety herein. Moreover, this technique is also described in U.S. Pat. Nos. 5,658,570, 5,693,780 and 5,756,096; each of which is incorporated herein by reference.
Once a monoclonal antibody has been identified (e.g. either a hybridoma-derived monoclonal antibody or a recombinant antibody from a combinatorial library, including monoclonal antibodies that are already known in the art), DNAs encoding the light and heavy chains of the monoclonal antibody are isolated by standard molecular biology techniques. For hybridoma derived antibodies, light and heavy chain cDNAs can be obtained, for example, by PCR amplification or cDNA library screening. For recombinant antibodies, such as from a phage display library, cDNA encoding the light and heavy chains can be recovered from the display package (e.g. phage) isolated during the library screening process. Nucleotide sequences of antibody light and heavy chain genes from which PCR primers or cDNA library probes can be prepared are known in the art. For example, many such sequences are disclosed in Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 and in the ″Vbase″ human germline sequence database.
Once obtained, the antibody light and heavy chain sequences are cloned into a recombinant expression vector using standard methods. As discussed above, the sequences encoding the hydrophobic leaders of the light and heavy chains are removed and sequences encoding a nuclear localization signal (e.g. from SV40 Large T antigen) are linked in-frame to sequences encoding either the amino- or carboxy terminus of both the light and heavy chains. The expression vector can encode an intracellular antibody in one of several different forms. For example, in one embodiment, the vector encodes full-length antibody light and heavy chains such that a full-length antibody is expressed intracellularly. In another embodiment, the vector encodes a full-length light chain but only the VH/CH1 region of the heavy chain such that a Fab fragment is expressed intracellularly.
In another embodiment, an inhibitory agent for use in the methods of the invention is a peptidic compound derived from the amino acid sequence of a negative regulator of intracellular levels of glucose, pyruvate, lactate, and/or NADPH.
Peptidic compounds useful in the method of the invention can be made intracellularly in cells by introducing into the cells an expression vector encoding the peptide. Such expression vectors can be made by standard techniques using oligonucleotides that encode the amino acid sequence of the peptidic compound. The peptide can be expressed in intracellularly as a fusion with another protein or peptide (e.g. a GST fusion). Alternative to recombinant synthesis of the peptides in the cells, the peptides can be made by chemical synthesis using standard peptide synthesis techniques.
Synthesized peptides can then be introduced into cells by a variety of means known in the art for introducing peptides into cells (e.g. liposome and the like).
Another form of an inhibitory agent which inhibits a negative regulator of the generation and/or uptake of glucose, pyruvate, lactate, and/or NADPH in a cell is a chemical small molecule compound.
This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures, are hereby incorporated by reference.
EXAMPLES
Example 1
Assessing Stimulation of the Insulin/mTOR Pathway and Resulting Effects on Cone Cell Death in a Mouse Model of Retinitis Pigmentosa
Material and Methods
Animals: Wild type (wt) mice (C57B1/6N) and PDE-β−/− mice (referred as rd1 or FVB/N) were purchased from Taconic Farms. The PDE-β−/− mice have a mutation in the β-subunit of cGMP phosphodiesterase (Bowes, C. et al. (1990) Nature 347, 677-80) (PDE). The PDE-γknock-out (PDE-γ-KO) lacks the γ-subunit of PDE (Tsang, S. H. et al. (1996) Science 272, 1026-9). The rhodopsin knock-out (Rho-KO) lacks the rod-specific opsin gene (Tsang, S. H. et al. (1996) Science 272, 1026-9; Lem, J. et al. (1999) Proc Natl Acad Sci USA 96, 736-41). The P23H mouse has a proline-23 to histidine mutation in the rhodopsin gene (Naash, M. I., et al. (1993) Proc Natl Acad Sci USA 90, 5499-503). As this mouse carries a transgene the strain was always crossed back to C57B1/6N to ensure that none of the progeny would carry two alleles of the transgene. The transgene is specifically expressed in rods (Gouras, P., et al. (1994) Vis Neurosci 11, 1227-31; Woodford, B. J., et al. (1994) Exp Eye Res 58, 631-5; al-Ubaidi, M. R. et al. (1990) J Biol Chem 265, 20563-9) and carries 3 mutations in the rhodopsin gene (Val-20 to Gly, Pro-23 to H is, Pro-27 to Leu). In this study it is referred as the P23H mutant. The cone-lacZ strain has been previously described (Wang, Y. et al. (1992) Neuron 9, 429-40). All procedures involving animals were in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Affymetrix array analysis: RNA was extracted as described previously (Punzo, C. & Cepko, C. (2007) Ophthalmol Vis Sci 48, 849-57). Three to 4 retinae were used per extraction. A minimum of two arrays were analyzed per time point. The statistical significance of each gene expression profile was determined by a Jonckheere-Terpstra test of the hypothesized cone-death patterned alternative, using exact p-values calculated by the Harding algorithm (Harding, E. F. (1984) Applied Statistics 33, 1-6).
qRT-PCR was performed as described previously with the same primers and conditions for Opn1sw and gapdh (Punzo, C. & Cepko, C. (2007) Ophthalmol Vis Sci 48, 849-57). The following primers and conditions were used for the three LAMP-2 splice forms: LAMP-2 forw. ctgaaggaagtgaatgtctacatg (SEQ ID NO:1); LAMP-2A rev. gctcatatccagtatgatggc (SEQ ID NO:2); LAMP-2B rev. cagagtctgatatccagcatag (SEQ ID NO:3); LAMP-2C rev. gacagactgataaccagtacg (SEQ ID NO:4). Conditions for all three PCRs: 95° for 3 sec, 52° for 15 sec, 72° for 25 sec. The data in FIG. 3 a and FIG. 13 d represent an average of 3 measurements corrected for gapdh.
Retinal explant cultures: The retina was dissected free from other ocular tissues in DMEM, and then incubated in conditions according to the chart in FIG. 10 a . Regular DMEM was at 4.5 g/L glucose, low glucose was at 1 g/L, leucine was added at 200 μM and FCS at 10%. Incubation was performed for 4 h and the retinae were fixed and processed for antibody staining as described below.
TUNEL, X-gal histochemistry and In Situ Hybridizations were performed as described previously (Punzo, C. & Cepko, C. (2007) Ophthalmol Vis Sci 48, 849-57). For the double labeling of cones (see FIG. 16 ), retinae were first fixed in 2% PFA for 15 min. then processed for the X-GAL reaction and then post fixed in 4% PFA for 15 min. A biotin-PNA was used in an antibody staining procedure (see below) and detected with Streptavidin-POD (1:500, Roche) by a DAB stain (Sigma) according to the manufacture's instructions. The following ESTs were used for the red/green opsin and blue opsin probes respectively: red/green opsin (BE950633); blue opsin (BI202577). Probe for rhodopsin was generated by sub-cloning the coding sequence of the gene into pGEM-T Easy (Promega). The following primers were used for amplification of the coding sequence: forw. agccatgaacggcacagaggg (SEQ ID NO:5); rev. cttaggctggagccacctggct (SEQ ID NO:6). The antisense RNA was generated with T7 RNA polymerase.
Viral injections were performed as described previously (Punzo, C. & Cepko, C. L. (2008) Dev Dyn 237, 1034-42). Mice were injected at embryonic day 10 and harvested at postnatal week 10. The fusion protein was generated with a NotI site at the 5' end followed by GFP, then LC3, and then an XhoI site at the 3' end and cloned into pQCXIX (Clonetech: cat. #631515). The following primers were used for the fusion protein: 5'NotI-GFP atgcgggccgccaccatggtgagcaagggcgaggagc (SEQ ID NO:7), 3'GFP-LC3 aggtcttctcggacggcatcttgtacagctcgtccatgc-cgag (SEQ ID NO:8), 5'LC3 atgccgtccgagaagaccttcaagc (SEQ ID NO:9), 3'LC3-XhoI atctcgagttacacagccattgctgtcccgaatg (SEQ ID NO:10).
Rapamycin, Streptozotocin and Insulin treatments were performed as follows. Rapamycin was diluted to 10 mg/ml in ethanol. The stock was diluted to 0.015 mg/ml in drinking water over a period of 2 weeks. A single intraperitoneal injection of 150 μl (12 mg/ml in 0.1M citric acid, ph4.5) of Streptozotocin was injected at postnatal day (P) 21. Insulin was injected intraperitoneally daily starting at P21. The concentration was increased weekly such that the first week, 10 U/kg body weight, the second 15 U/kg, the third 20 U/kg and fourth 30 U/kg body weight, were injected. In the treatment that lasted 7 weeks 30 U/kg body weight were injected for the remaining 3 weeks. Blood glucose levels were measured by collecting a drop of blood from the tail directly onto a test strip from TrueTrack smart system (CVS pharmacy). Eye bleeds were avoided due to the fact that cell survival in the retina was being assayed.
Quantification of cone survival was performed as follows. The colors of the bright light image were inverted and processed with Imaris software (Bitplane Inc) to calculate the percentage of blue surface area versus the total retinal surface area (see also FIG. 17 ). A minimum of 8 retinae per treatment, and for the control, were analyzed. P-values were calculated by the student's t-test. The cone lacZ transgene was chosen over PNA as a cone marker since the transgene labels cones more persistently, since, due to the shortening of the cone OS, PNA was found to stain less reliably than lacZ (see FIG. 16 ).
Whole mount and section antibody staining were performed as previously (Punzo, C. & Cepko, C. L. (2008) Dev Dyn 237, 1034-42) described with the following modifications. Antibody staining for LAMP-2: Triton was replaced with 0.01% Saponin. Antibody staining for p*-mTOR and p*-S6: PBS was replaced by TBS in every step of the procedure. Primary antibody dilutions: mouse α-rhodopsin Rho4D21:20051; goat α-β-Galactosidase (Serotec) 1:400; rabbit α-blue opsin (Jeremy Nathans) 1:1000; rabbit α-Gnat1 1:200 (Santa Cruz); rabbit α-Cleaved Caspase-3 (Cell Signaling) 1:100; rabbit α-Cleaved Lamin A (Cell Signaling) 1:100; rabbit α-GLUT-1 (Alpha Diagnostics) 1:100; rabbit α-p*-mTOR (Ser2448) (Cell Signaling) 1:300; rabbit α-p*-S6 (Ser235/236) (Cell Signaling) 1:100; rabbit α-HIF-1α (R&D Systems) 1:300; rat α-LAMP-2 (clone: GL2A7, from DSHB) 1:200. Time points analyzed for the rod and cone death kinetics (P: postnatal day; PW: postnatal week): PDE-β−/−: P10-P20 daily, PW3-10 weekly, PW 12, PW15, PW18, PW45; PDE-γ-KO: P10-P20 daily, PW3-PW10 weekly, PW15, PW25, PW45; Rho-KO: PW4-PW8 weekly, PW10, PW11, PW17, PW20, PW25, PW27, PW31, PW34, PW37, PW45, PW55, PW80; P23H: PW5, PW10, PW16, PW25, PW30, PW35, PW40, PW65, PW70, PW75, PW80, PW85.
Results
Rod and Cone Death Kinetics
To establish a framework for comparing gene expression in 4 different models of RP, the equivalent stages of disease pathology were established through examination of the kinetics of rod ( FIG. 1 ) (see also FIGS. 2 and 4 ) and cone ( FIG. 3 ) (see also FIG. 6 ) death. Rod death kinetics were established by determining the onset, progression and end phase of rod death ( FIG. 1 ). The time from the onset of rod death to the time when the outer nuclear layer (ONL) was reduced to 1 row of cells will be referred to as the major rod death phase. The time thereafter until rod death was complete will be referred to as the end phase of rod death. To determine the beginning of the major phase of rod death, cleavage of the nuclear envelope protein LaminA ( FIG. 1 a ), and of the apoptotic protease Caspase3 ( FIG. 1 b ), as well as TUNEL ( FIG. 1 c, d ) were used. The continuation of the major rod death phase was monitored by these assays, as well as inspection of histological sections ( FIG. 1 e - h ), as rods account for more than 95% of all PRs. Once the ONL reached one row of cells, the major phase of rod death was over. The end phase of rod death was determined using rod-specific markers to perform either in situ hybridization (see FIG. 2 ) or immunohistochemistry ( FIG. 1 i - l ) on retinal sections. However, unless every section of a single retina is collected it is difficult to determine if any rods remain. Thus, retinal flat mounts also were used to allow a comprehensive analysis of the end phase of rod death ( FIG. 1 m - q ). Interestingly, while in the two PDE mutants and in the Rho-KO mutant the end phase of rod death was clearly defined, in the P23H mutant, rods died so slowly that even 50 weeks (latest time point analyzed) after the end of the major phase of rod death, some rods were still present (see FIG. 4 ).
Two methods were used to determine the onset and progression of cone death. First, the overall time frame of cone demise was determined by quantitative real-time polymerase chain reaction (qRT-PCR) ( FIG. 3 a ) for the ventral (Applebury, M. L. et al. (2000) Neuron 27, 513-23) cone specific transcript Opn1sw (opsin1 short-wave-sensitive: blue cone opsin). This allowed for an initial quantitative comparison among different strains, but was not adequate to determine the number of cones as transcript levels could vary prior to cell death. Next, whole mount immunohistochemistry for red/green opsin (Opn1mw: opsin1 medium-wave-sensitive) and peanut agglutinin lectin (PNA) were used ( FIG. 3 b - n ). Both markers are expressed throughout the murine retina allowing for the visualization of cones ( FIG. 3 b - d ). Interestingly, the onset of cone death always occurred at the equivalent stage of rod death, namely after the major rod death phase, when the thickness of the ONL was reduced to only a single row of cells. Cone death was found to proceed from the center to the periphery in all 4 models, as seen by staining with PNA ( FIG. 3 e ). It was preceded by a gradual reduction of the outer segment (OS) length ( FIG. 3 f - i ) and by opsin localization from the OS to the entire cell membrane ( FIG. 3 j - l ). In addition, red/green opsin (Opn1mw) protein, which is normally detected throughout the mouse retina ( FIG. 3 b ), was detected mainly dorsally during cone degeneration ( FIG. 3 m , n). However, PNA staining showed no appreciable difference across the dorsal/ventral axis ( FIG. 3 m, n ). Similarly, blue opsin expression, which is normally detected only ventrally (Applebury, M. L. et al. (2000) Neuron 27, 513-23) ( FIG. 3 c, d ), was not affected during degeneration ( FIG. 3 o ). Shortening of cone OSs and loss of cone-specific markers has also been described in human cases of RP (John, S. K., et al. (2000) Mol Vis 6, 204-15).
In summary, the kinetics and histological changes that accompanied rod and cone death shared several features across the 4 models. First, cone degeneration always started after the major rod death phase ( FIG. 5 a, b ). This point was reached at very different ages in three of the 4 mutants, as the overall kinetics of rod death were quite different. Second, cone death was always central to peripheral and was preceded by a reduction in OS length. Third, in all 4 mutants, red/green opsin protein levels were detectable mainly dorsally during cone degeneration ( FIG. 5 c ). These common features evidence a common mechanism(s) of cone death. Moreover, gene expression changes that were common across the 4 models at the onset of cone death serve to elucidate this common mechanism.
Microarray Analysis
To determine common gene expression changes, RNA samples from all 4 models were collected halfway through the major phase of rod death, at the onset of cone death, and from two time points during the cone death phase ( FIG. 7 a ). The RNA was then hybridized to an Affymetrix 430 2.0 mouse array. Gene expression changes were compared within the same strain across the 4 time points. Two criteria had to be fulfilled to select a gene for cross comparison among the 4 strains. First, the change over time had to be statistically significant (see Material & Methods). Second, a gene had to be upregulated at least 2 fold at the onset of cone death compared to the other three time points. This second criterion removed rod-specific changes that were still occurring at the onset of cone death while at the same time enriched for changes at the onset of cone death. A total of 240 Affymetrix IDs were found that satisfied both criteria within each of the 4 strains. The 240 IDs matched to 230 genes (see FIGS. 19A-E ). Of the 195 genes that could be annotated, 34.9% (68 genes) were genes involved in cellular metabolism ( FIG. 7 b, c ). The signaling pathway with the highest number of hits (12 genes) was the insulin/mTOR (mammalian target of rapamycin) signaling pathway ( FIG. 7 b ), a key pathway in regulating many aspects of cellular metabolism. Thus, the data evidences that events at the onset of cone death coincided with changes in cellular metabolism likely to be regulated by the insulin/mTOR pathway.
mTOR in Wild Type and Degenerating Retinae
Based on the findings of the microarray analysis, the insulin/mTOR signaling pathway was examined during the period of cone death. The kinase, mTOR, is a key regulator of protein synthesis and ribosome biogenesis (Reiling, J. H. & Sabatini, D. M. (2006) Oncogene 25, 6373-83). When cellular energy levels are high, mTOR allows energy consuming processes, such as translation, and prevents autophagy, while nutrient poor conditions have the reverse effect. Therefore, glucose, which increases cellular ATP levels, and amino acid availability, especially that of leucine, positively affect mTOR activity. To understand if cellular energy levels or amino acid availability might be compromised in cones during degeneration, levels of phosphorylated mTOR (p*-mTOR) were examined by immunofluorescence. Phosphorylation of mTOR increases kinase activity, and therefore levels of p*-mTOR can serve as an indicator of its activity level. Since every eukaryotic cell expresses mTOR, a certain level of p*-mTOR is likely to be found in every cell. Surprisingly, high levels of p*-mTOR were detected only in dorsal cones of wild type retinae ( FIG. 9 a - c ). This phosphorylation pattern was reminiscent of the red/green opsin pattern seen during cone degeneration ( FIG. 5 c ). Since mTOR is a key regulator of translation, we investigated whether the ventral red/green opsin downregulation that occurred during cone degeneration could be mimicked by a reduction in mTOR activity. To this end, wild type mice were treated with rapamycin, an mTOR inhibitor18. This treatment resulted in ventral downregulation of red/green opsin, without affecting blue opsin or PNA staining or the dorsal phosphorylation of mTOR itself ( FIG. 9 d - g ). Thus, inhibition of mTOR in wild type recapitulated the expression of red/green opsin and blue opsin, as well as the pattern of PNA staining, in the mutants during degeneration, indicating that the ventral downregulation of red/green opsin seen during degeneration might be due to reduced mTOR activity. As expected for mTOR function, the downregulation of red/green opsin did not occur at the RNA level, but at the protein level, in untreated mutant mice, as well as in wild type mice treated with rapamycin (see FIG. 8 ). Finally, analysis of mutant retinae showed a decrease of p*-mTOR levels in dorsal cones during cone degeneration ( FIG. 9 h - m ). To test whether the high level of p*-mTOR found in dorsal wild type cones was glucose-dependent, retinal explants of wild type mice were cultured in media for 4 hours in the presence or absence of glucose. Dorsal p*-mTOR was abolished in the absence of glucose even when leucine concentrations were increased in the medium (see FIG. 10 ). Thus, the data on mTOR establish a link between mTOR activity, the expression changes of red/green opsin seen during degeneration, and the microarray data, which indicated metabolic changes at the onset of cone death. Those changes may be caused by compromised glucose uptake in cones.
Responses of Cones to Nutritional Imbalance
The data on mTOR evidenced a nutritional imbalance in cones during cone degeneration, possibly caused by reduced glucose levels in cones. To test this idea, the level of the heterodimeric transcription factor, Hypoxia inducible factor 1 (HIF-1α/β), which improves glycolysis under stress conditions such as low oxygen, was examined. HIF-1 and mTOR are tightly linked as low oxygen results in low energy due to reduced oxidative phosphorylation, and therefore in reduced mTOR activity (Reiling, J. H. & Sabatini, D. M. (2006) Oncogene 25, 6373-83; Dekanty, A., et al. (2005) J Cell Sci 118, 5431-41; Hudson, C. C. et al. (2002) Mol Cell Biol 22, 7004-14; Treins, C., et al. (2002) J Biol Chem 277, 27975-81; Zhong, H. et al. (2000) Cancer Res 60, 1541-5; Thomas, G. V. et al. (2006) Nat Med 12, 122-7). An upregulation of the regulated subunit HIF-1α would likely reflect low glucose levels in cones, and not hypoxic conditions, as oxygen levels are increased due to the loss of rods (Yu, D. Y. & Cringle, S. J. (2005) Exp Eye Res 80, 745-51). Immunofluorescence analysis of HIF-1α during cone degeneration revealed an upregulation of the protein in cones in all 4 mouse models ( FIG. 11 a - f and 12 a - d ). Consistent with the upregulation of HIF-1α, glucose transporter 1 (GLUT1), a HIF-1α target gene (Wang, G. L., et al. (1995) Proc Natl Acad Sci USA 92, 5510-4; Ebert, B. L., et al. (1995) J Biol Chem 270, 29083-9) also was found to be upregulated in cones, again in all 4 mouse models ( FIG. 11 g - j and FIG. 12 e - h ). Thus HIF-1α and GLUT1 upregulation are consistent with a response in cones to overcome a shortage of glucose. It also provides a link to the decreased p*-mTOR levels found during degeneration as well as the sensitivity of p*-mTOR to glucose.
To ascertain if cones are nutritionally deprived, autophagy within cones was assessed. Two types of autophagy are inducible by various degrees of nutrient deprivation: macroautophagy and chaperone mediated autophagy (CMA) (Massey, A., et al. (2004) Int J Biochem Cell Biol 36, 2420-34; Finn, P. F. & Dice, J. F. (2006) Nutrition 22, 830-44; Codogno, P. & Meijer, A. J. (2005) Cell Death Differ 12 Suppl 2, 1509-18; Dice, J. F. (2007) Autophagy 3, 295-9). Macroautophagy is non-selective, targets proteins or entire organelles, and is marked by de novo formation of membranes that form intermediate vesicles (autophagosomes) that fuse with the lysosomes. The machinery required for macroautophagy has been shown to be present in PRs (Kunchithapautham, K. & Rohrer, B. (2007) Autophagy 3, 433-41). In contrast, CMA is selective and targets individual proteins for transport to the lysosomes. The presence of macroautophagy was assessed by infection with a viral vector encoding a fusion protein of green fluorescent protein (GFP) and light chain 3 (LC3), an autophagosomal membrane marker (Kabeya, Y. et al. (2000) Embo J 19, 5720-8; Mizushima, N., et al. (2004) Mol Biol Cell 15, 1101-11; Punzo, C. & Cepko, C. L. (2008) Dev Dyn 237, 1034-42). No difference was observed in GFP distribution in cones of wild type and mutant mice, indicating that formation of autophagosomes was absent during cone death (see FIG. 14 a - f ). Additionally, high levels of phosphorylated ribosomal protein S6 were found in all, or most, cones (see FIG. 14 g - h ) reflecting an increased activity of ribosomal S6 kinase 1 (S6K1), an inhibitor of macroautophagy (Codogno, P. & Meijer, A. J. (2005) Cell Death Differ 12 Suppl 2, 1509-18). Consistent with these findings is the fact that macroautophagy reflects an acute short-term response to nutrient deprivation or cellular stress conditions (Massey, A., et al. (2004) Int J Biochem Cell Biol 36, 2420-34; Finn, P. F. & Dice, J. F. (2006) Nutrition 22, 830-44). Prolonged non-selective degradation of newly synthesized proteins to overcome the stress condition would not be favorable to cells and would likely result in the relatively rapid death of most cones, rather than the slow death seen in RP.
CMA is normally activated over extended periods of starvation and results in increased levels of lysosomal-associated membrane protein (LAMP) type 2A at the lysosomal membrane (Massey, A., et al. (2004) Int J Biochem Cell Biol 36, 2420-34; Finn, P. F. & Dice, J. F. (2006) Nutrition 22, 830-44; Cuervo, A. M. & Dice, J. F. (200) Traffic 1, 570-83). Both starvation and oxidative stress can induce CMA (Massey, A., et al. (2004) Int J Biochem Cell Biol 36, 2420-34). Starvation increases LAMP-2A by preventing its degradation while oxidative stress results in de novo synthesis of LAMP-2A (Kiffin, R., et al. (2004) Mol Biol Cell 15, 4829-40). A LAMP-2 antibody that recognizes the proteins resulting from all 3 splice isoforms (Cuervo, A. M. & Dice, J. F. (2000) J Cell Sci 113 Pt 24, 4441-50) (A, B, C) showed high levels of LAMP-2 at the lysosomal membrane in all 4 mutants during cone degeneration ( FIG. 13 a - c ; data only shown for PDE-β−/−). The high levels were specific to cones and were not seen in cells of the inner nuclear layer ( FIG. 13 b, c ), which might reflect the possibility that cones are the only starving cells in the RP retina. qRT-PCR for the three splice isoforms showed only a minor increase in mRNA levels of LAMP-2A (1.2×) and a decrease in LAMP-2C ( FIG. 13 d ) indicating that the increase seen in protein at the membrane is mainly due to nutritional deprivation and only to a lesser extent to oxidative stress (Komeima, K., et al. (2006) Proc Natl Acad Sci USA 103, 11300-5; Komeima, K., et al. (2007) J Cell Physio 213, 809-815; Kiffin, R., et al. (2004) Mol Biol Cell 15, 4829-40). Taken together, the data demonstrates that nutritional imbalance in cones leads to the activation of CMA, a process that is consistent with prolonged starvation.
Stimulation of the Insulin Receptor Pathway Prolongs Cone Survival
The data on mTOR, HIF-1α, GLUT1 and the induction of CMA demonstrated that a shortage of glucose in cones resulting in starvation and further demonstrated that the insulin/mTOR pathway plays an important role during cone death. To determine if the insulin/mTOR pathway can influence cone survival, we stimulated the pathway by systemic treatment of PDE-β−/− mice with insulin. The PDE-β mutant was chosen over the other three mutants due to its faster cone death kinetics, allowing for a better read-out of cone survival. Mice were treated with daily intraperitoneal injections of insulin over a 4 week period, starting at the onset of cone death. To reduce insulin, a single injection of streptozotocin, a drug that kills the insulin-producing beta cells of the pancreas, also was examined. Systemic administration of insulin results in a desensitized insulin receptor due to a feedback loop in the pathway, which causes an increase in blood glucose levels. Injection of streptozotocin, which also results in increased blood glucose levels, served as a control for the effect of elevated blood glucose, and also provided animals with reduced levels of insulin. PDE-β−/− mice injected with insulin showed improved cone survival compared to uninjected control mice. PDE-β−/− mice injected with Streptozotocin showed a decrease in cone survival ( FIG. 15 a - d ). Improved cone survival was therefore due to insulin and not to the increased blood glucose levels ( FIG. 15 e ). Additionally, cones in mutant mice treated with insulin did not show the upregulation of HIF-1α seen normally in cones during degeneration, consistent with the notion that cones were responding to insulin directly ( FIG. 15 g, h ).
Discussion
The results presented herein show that cones exhibit signs of nutritional imbalance during the period of cone degeneration in RP mice. The microarray analysis demonstrates that there are changes in cellular metabolism involving the insulin/mTOR pathway at the onset of cone death. It was demonstrated that inhibition of mTOR in wild type mice resulted in the same pattern of loss of red/green opsin as seen during degeneration. In accord with changes in p*-mTOR, and its sensitivity to glucose, an upregulation of HIF-1α and GLUT1 was observed, demonstrating that glucose uptake, and/or the intracellular levels of glucose, may be compromised in cones of RP mice. Additionally, systemic administration of insulin prolonged cone survival, whereas depletion of endogenous insulin had the reverse effect. The systemic treatment with insulin prevented the upregulation of HIF-1α in cones seen normally during cone degeneration, demonstrating that insulin was directly acting on cones. Interestingly, a prolonged treatment of insulin during a time span of 7 weeks instead of 4 weeks did not show any significant improvement of cone survival (see FIG. 18 ). This may reflect the feedback loop of the pathway in which S6K1 acts directly onto the insulin-receptor substrate (IRS). The results indicate that nutrient availability in cones may be altered during the period of cone degeneration and that the insulin/mTOR pathway plays a crucial role. A recent report showed that constitutive expression of proinsulin in the rd10 mouse model of RP delays photoreceptor death, both of rods and cones (Corrochano, S. et al. (2008) Invest Ophthalmol Vis Sci 49, 4188-94). However, proinsulin seems not to act through the insulin receptor as mice treated with proinsulin did not develop hyperglycemia. Proinsulin blocks developmental cell death and thus may interfere with the apoptotic pathway in the postnatal retina. Macroautophagy, which is controlled by mTOR through its downstream target S6K1, was not detected during cone degeneration, while CMA appeared to be activated. Increased LAMP-2A levels at the lysosomal membrane indicated activation of CMA. In addition, the observations concerning mTOR, HIF-1α, and GLUT1 are consistent with starvation and CMA. The lack of detectable macroautophagy does not rule out the possibility that macroautophagy might occur for a short period of time (e.g., 24 hours) prior to the activation of CMA. The data only show that macroautophagy is not the main form of autophagy over an extended period of time, which is consistent with the notion that macroautophagy is a short-term response. The prolonged inhibition of macroautophagy is likely due to increased S6K1 activity as seen by increased p*-S6 levels. S6K1 is positively regulated by mTOR and AMP-activated protein kinase (AMPK) (Codogno, P. & Meijer, A. J. (2005) Cell Death Differ 12 Suppl 2, 1509-18), which reads out cellular ATP levels. Therefore, while mTOR may report metabolic problems with respect to glucose uptake, and reduce energy consuming processes and improve glycolysis through HIF-1α, AMPK may report normal cellular ATP levels and inhibit macroautophagy. This represents a specific response to the energy requirements of cones. Most of the glucose taken up by PRs never enters the Krebs cycle (Poitry-Yamate, et al. (1995) J Neurosci 15, 5179-91). Thus the shortage of glucose may not cause a shortage of ATP. Lactate, provided by Muller glia, can generate ATP via the Krebs cycle (Tsacopoulos, M., et al. (1998) Prog Retin Eye Res 17, 429-420. However, glucose is needed to generate NADPH in the pentose phosphate cycle, and NADPH is required for synthesis of phospholipids, the building blocks of cell membranes. PRs constantly shed their membranes at the tip of the OSs. Since reduced levels of glucose would result in reduction of membrane synthesis, the rate of OS phagocytosis by the RPE may be higher than the rate of membrane synthesis by cones. Consistent with this, OS shortening preceded cell death in these 4 models, as is also observed in human cases of RP17. Additionally, changes that affect lipid metabolism were also seen by the microarray analysis.
These studies described herein were designed to determine why the loss of rods result in cone death in RP. The previous hypotheses attributing cone death either to a toxin released by rod cells or to the lack of a trophic factor produced by rod cells and necessary for cone survival each fail to explain the pathology found in humans. The rod and cone death kinetics shown here clearly argue against a toxin produced by dying rods as a cause for cone death since the onset of cone death always occurred after the major rod death period. If a rod toxin caused cone death, then the onset of cone death should have either coincided with the onset of rod death or should have started shortly thereafter, since this would be the period of peak toxin production. Interestingly, the lack of a trophic factor produced by healthy rods and required for cone survival would agree with the onset of cone death seen in all four models as one would expect the onset of cone death during the end stages of rod death. However, the progression of cone death and the end phase of rod death make this unlikely hypothesis as the sole reason for cone death. In the two PDE mutants and in the Rho-KO mutant, cones were dying for many weeks after the end phase of rod death, indicating that they could survive quite awhile in the absence of rods. In addition, in the P23H model, rods died so slowly during the end phase of rod death, that during the entire period of cone death, rods were still present. The hypothesis that a lack of a rod trophic factor being the main cause for cone death seems unlikely given these discrepancies.
Our observations of nutritionally deprived cones demonstrate the dependence of cones on rods. The OS-RPE interactions are vital since the RPE shuttles nutrition and oxygen from the choroidal vasculature to PRs. Roughly 95% of all PRs in mouse and human are rods and approximately 20-30 OSs contact one RPE cell (Snodderly, D. M., et al. (2002) Invest Ophthalmol Vis Sci 43, 2815-8; Young, R. W. (1971) Journal of Cell Biology 49, 303-318). Thus, only 1-2 of those RPE-OS contacts are via cones. During the collapse of the ONL, the remaining cone:RPE interactions are likely perturbed. If these interactions drop below a threshold required for the proper flow of nutrients, the loss of rods results in a reduced flow of nutrients to cones. In all 4 mouse models, the onset of cone death occurred when the ONL reached one row of cells. This cell density therefore represents the critical threshold. Then, while the remaining rods die due to a mutation in a rod-specific gene, cone death begins due to nutrient deprivation. In accord with this notion, cone death progressed more slowly when the remaining rods died slowly. This mechanism would also explain why the loss of cones does not lead to rod death (Biel, M. et al. (1999) Proc Natl Acad Sci USA 96, 7553-7; Yang, R. B. et al. (1999) J Neurosci 19, 5889-970. Since in humans and mouse, cones are less than 5% of all PRs, the critical threshold that perturbs OS-RPE interactions would not be reached. Further support for this idea is provided by studies in zebrafish where the overall ratio of rods to cones is reversed (1:8). Additionally, the distribution of rods and cones in zebrafish is uneven such that certain regions are cone-rich whereas other regions are rod-rich. A recently isolated mutation in a cone-specific gene resulted in rod death, but only in regions of high cone density (Stearns, G., et al. (2007) J Neurosci 27, 13866-74), leading Stearns and co-workers to conclude that cell density is the crucial determinant. We determined that once a critical threshold of cell density is breached, improper OS-RPE interactions result in reduced flow of nutrients (e.g., glucose). This results in reduced OS membrane synthesis, which in turn further contributes to a reduced uptake of nutrients from the RPE. Ultimately, prolonged starvation, as indicated by the activation of CMA, leads to cell death. Since starvation can occur slowly over extended periods of time, and because the rate may fluctuate due to fluctuations in nutrient uptake, the slow and irregular demise of cones observed in humans results therefrom. Therefore, the results presented herein not only provide a new mechanism of cone death in RP that should direct future therapeutic approaches, but also consolidate the data from the literature with respect to the death kinetics of rods and cones seen in mice and patients with different RP mutations.
Example 2
Transfection of Cone Cells with Gluconeogenic Genes to Increase Cone Viability
Neurons can be compromised by genetic and environmental factors that lead to their malfunction and death. In the retina, specialized sensory neurons, the photoreceptors (rods and cones), as well as ganglion cells, the output neurons of the retina, are the neuronal cell types that malfunction and die, leading to partial or complete loss of vision. The reasons that retinal neurons die in a disease, Retinitis Pigmentosa, that leads to loss of vision have been investigated. In Retinitis Pigmentosa, mutation of a gene that is only expressed in rods leads to the death of the rods. Subsequently, cones also die. It has been discovered that cones suffer from a lack of activity in the mTOR pathway, and appear to be starving. Starvation in cones was indicated by the activation of the chaperone mediated autophagy pathway, whereby a cell digests selected proteins when under starvation conditions. Prior to autophagy, it was noted that the cone outer segments were shrinking, and that the synthesis and/or turnover of red/green opsin protein led to reduction of this protein in cones. These observations led to the determination that cones were starving due to a lack of glucose. It was reasoned that membrane biosynthesis was slowed, leading to a reduction in the size of the outer segments, which are very membrane rich. Membrane biosynthesis requires acetylCoA, which is derived from glucose (as well as other molecules). In addition, membrane biosynthesis, as well as many other anabolic reactions, require NADPH, which can be generated by the pentose phosphate pathway, which originates with glucose. The reduction in red/green opsin protein levels also was consistent with lack of a nutrient(s) as it could be due to lack of robust translation, and/or rapid turnover of this protein. In keeping with the rationale of insufficient glucose was a lack of detectable phosphorylation of mTOR in cones. It was shown that mTOR phosphorylation in cones was dependent upon glucose. Lack of mTOR phosphorylation leads to a reduction in translation and, thus, reduction in red/green opsin, as well as other proteins. Finally, the autophagy was also consistent with a reduction in nutrients, and glucose is a key nutrient. Therefore methods to supply cones with more glucose, and/or more NADPH were investigated.
Two ways were used to supply cells with more glucose. One was to increase intracellular glucose levels by providing cones with the means to synthesize their own glucose. Glucose synthesis, called gluconeogenesis, is carried out primarily in the liver, and to a limited extent in kidney and muscle. One might consider that the enzymes required for glucose synthesis could be those that break down glucose, during the process of glycolysis. However, some of the steps in glycolysis are energy producing and thus to reverse them requires a different process. Three enzymes must be supplied to allow the glucose to be synthesized, utilizing pyruvate or lactate as a starting point, and utilizing ATP and GTP to go uphill energetically in the synthesis. The first energy requiring step is catalyzed by Pcx (Pcx: Pyruvate Carboxylase) and utilizes ATP. A subsequent energy requiring step is catalyzed by Pck1: Phosphoenolpyruvate carboxykinase) and requires GTP. The third gene that is required to carry out gluconeogenesis is Fbp1 (Fructose 1,6 biphosphatase). Both Pck1 and Fbp1 have two isoforms, known as Pck2 and Fbp2. Pck1 and Fbp1 are expressed in the liver and are located in the cytoplasm whereas Pck2 and Fbp2 are located in the mitochondria and are expressed normally in muscle cells. To allow cone cells to synthesize their own glucose, cones were infected with an AAV virus (AAV 2/5; genome from serotype 2, with rep and cap genes from serotype 5) that carries these 3 genes. The AAV2/5 has the advantage that it infects or expresses in cones. The vector design is as follows: CMV promotor-Pcx-IRES-Fbp1-IRES-Pck1 (see FIGS. 21 and 29 ; the sequence of the vector depicted in FIG. 29 is set forth in SEQ ID NO:11). The coding sequences for all three genes are on one transcript and the IRES elements (Internal ribosomal entry sites) allows translation of the two genes (Fbp1 and PCk1) downstream of Pcx. The endogenous expression of Fbp1, PCk1, and Pcx and the expression Fbp1, PCk1, and Pcx in the AAV vector (“construct”) was analyzed by PCR analysis ( FIG. 22A ) and Western blot analysis ( FIG. 22B ).
This vector was tested in vivo in the rd1 mutant mouse, which has shown additional cone survival relative to controls. This result confirms that cones are starving due to limiting glucose. The morphology of the cone outer segments is remarkably similar to that of normal cones, with robust inner and outer segments, with red/green opsin properly localized to the outer segment. This observation also confirms that outer segment shortening was due to insufficient glucose. The animals were also tested in two behavioral assays, with infected animals demonstrating functional vision (see FIGS. 23 and 24 ).
An additional strategy to supply cones with more glucose is to provide them with a gene encoding a glucose transporter, such as glut1. The gene encoding glut1 is delivered using an AAV2/5 vector. There is likely excess extracellular glucose in the region around the cones. Rods constitute 97% of the cells in the area occupied by cones and, thus, the glucose that would have been taken up by the rods should still be available after the rods die. Cones with additional glut1 are able to take up more of this additional glucose. Another way to supply more nutrition to cones is to boost the uptake of nutrients from the retinal glial cell type that is in intimate contact with photoreceptors (the Muller glial cells). Muller glial cells also take up glucose and can use the glucose to produce, and then release, lactate, and perhaps pyruvate. The lactate can then be taken up by photoreceptors through a transporter, such as MCT1 or MCT2. In order to boost this process, the glut 1 gene can be delivered to Muller glia and/or the MCT1 or MCT2 gene can be delivered to photoreceptors. Finally, NADPH may be the limiting molecule that causes cones to die. NADPH is used for anabolic processes, as well as for detoxifying free oxygen radicals. After the rods die, the oxygen that would have been consumed by them is in excess in the vicinity of the cones. Excess free oxygen and light, as well as the phototransduction process, can lead to more oxygen free radicals, which are damaging to cellular macromolecules. NADPH may be utilized primarily for this purpose, reducing the supply of NADPH for anabolic processes, such as membrane biosynthesis. To supply more NADPH, the gene encoding malic enzyme is supplied in an AAV2/5 vector. This enzyme catalyzes NADPH synthesis in the cytoplasm, using malate that originates from citrate that exits from the mitochondria.
Example 3
Transfection of Cone Cells with Gluconeogenic Genes to Increase Cone Viability
As described in Example 2, AAV vectors (2/5) were created to transmit the three gluconeogenesis genes, Pcx, Pck, and Fbp-1 (as a single vector comprising the three genes operably linked to the CMV promoter), and were used to produce glucose in transfected cells.
As described below, the three genes (Pcx, Pck, and Fbp-1) were separated into different AAV vectors operably linked to the cone-specific promoter, CAR, from the cone arrestin gene. In some cases, a gluconeogenesis gene was also operably linked to the marker gene, H2BGFP, a nuclear form of GFP, or mGFP, a membrane-bound form of GFP, to allow the tracking of infected cells. FIG. 30 depicts a map of the AAV2/5 vector comprising the CAR promoter, the gluconeogenesis gene, Pcx-1, and mGFP (the sequence of this vector is set forth in SEQ ID NO:12); FIG. 31 depicts a map of the AAV2/5 vector comprising the CAR promoter and the gluconeogenesis gene, Pck-1 (the sequence of the vector is set forth in SEQ ID NO:13); FIG. 32 depicts a map of the AAV2/5 vector comprising the CAR promoter, H2BGFP, and the gluconeogenesis genes, Fbp-1 and Pck-1 (the sequence of this vector is set forth in SEQ ID NO:14); FIG. 33 depicts a map of the AAV2/5 vector comprising the CAR promoter and the gluconeogenesis gene, Fbp-1 (the sequence of this vector is set forth in SEQ ID NO:15); FIG. 34 depicts a map of the AAV2/5 vector comprising the CAR promoter and the gluconeogenesis gene, Pcx-1 (the sequence of this vector is set forth in SEQ ID NO:16).
Alternatively, an AAV-GFP vector was co-injected with the three AAV vectors comprising a gluconeogeneis gene to allow detection of successful targeting of the viral inoculum. These vectors were used to infect the retina of wild type, CD1, mice to determine if protein encoded by the three gluconeogeneis gene was produced. Retinas were injected as described and subsequently used to make protein extracts for analysis by Western blot or for immunohistochemistry.
In one series of experiments, CD1 mice were injected at postnatal day 0 (P0) with three different viral vectors, each comprising a single gluconeogeneis gene operably linked to a CAR promoter (see, e.g., FIGS. 31, 33, and 34 ). Retinas were harvested at P32. The viral titer for injection was about 1×10 13 for each individual virus. A virus expressing GFP was also co-injected at low concentration (5×10 10 ) to identify the retinas with the best injections. Six retinas positive for GFP were processed for Western blot analysis. Each lane was loaded with 40 μg of cytoplasmic protein extract. As depicted in FIG. 25 , Western blotting analysis of protein extracts from retinas of CD1 mice transfected with the three virsus, each containing one of the gluconeogeneisis genes, Pcx, Fbp1 and Pck1, demonstrates that all three genes were overexpressed as compared to un-infected control retinas.
In another series of experiments, CD1 mice were injected at postnatal day 0 (P0) with three different viral vectors each comprising a single gluconeogeneis gene operably linked to a CAR promoter (see, e.g., FIGS. 31, 33, and 34 ) and retinas were harvested at P32. The viral titer for injection was about 1×10 13 for each individual virus. A virus expressing GFP was also co-injected at low concentration (5×10 10 ) to identify the retinas with the best injections. As shown in FIGS. 26 and 28 , immunofluorescence staining of the harvested retinas demonstrates overexpression of Pcx ( FIG. 26 ) and Fbp1 ( FIG. 28 ) in photoreceptors.
In addition, one AAV vector comprising a gluconeogeneis gene, Pcx, operably linked to the CMV promoter and mGFP (pAAV-CMVpq-Pcx-1-mGFP) and a seond AAV vector comprising the gluconeogeneis genes, Fbp1 and Pck1, operably linked to the CMV promoter and H2BGFP (pAAV-CMVpq-H2BGFP-1-Fbp1-Pck1) were injected into CD1 mice at P0 and retinas were harvested at P18. FIG. 27 shows selective expression of Pcx in cells that are also positive for mGFP demonstrating that the overexpression of Pcx in FIG. 26 is due to specific binding of the antibody.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.