CA2273173A1 - Extension of lifespan by overexpression of a gene that increases reactive oxygen metabolism - Google Patents

Abstract

The present invention provides a method for prolonging the lifespan of a cell by inserting into that cell a gene encoding a protein that increases reactive oxygen metabolism and expressing the gene. The gene is one that encodes the CuZn superoxide dismutase enzyme (SOD) and the cell is, in particular, a motorneuron. The invention also provides a method for increasing the lifespan of a human or animal, in particular companion animals and livestock, by inserting into the cells of the human or animal, a gene encoding SOD. In particular, the cells of the human or animal where the gene is inserted comprise motorneurons. The invention also extends to vectors for the expression of a SOD gene in a cell and to pharmaceutical compositions comprising the SOD gene or vectors comprising the SOD gene.

Description

Title: EXTENSION OF LIFESPAN BY OVEREXPRESSION OF A GENE THAT
INCREASES REACTIVE OXYGEN METABOLISM
FIELD OF THE INVENTION
The present invention is in the field of molecular genetics and transgenics.
In particular, the present invention provides a method of extending the lifespan of a human or animal by the overexpression of a gene that increases reactive oxygen metabolism in that human or animal.
1o BACKGROUND OF THE INVENTION
CuZn superoxide dismutase (SOD) catalyses the dismutation of the superoxide radical anion (Oz', reactive oxygen) to hydrogen peroxide and is a corner stone in the system of metabolic defenses that gives aerobic cells access to the benefits of oxidative metabolism [1, 2].
15 Enzymatically, SOD is unique in its structural stability and its catalytic dependence on electrostatic interactions.
As shown most vividly in yeast [3, 4] and Drosophila [5-7], structural mutations that nullify the catalytic activity of SOD can lead to complex and highly detrimental phenotypes arising from rampant and unchecked oxidative damage. Curiously, SOD mutations in mammals 2o are relatively benign from a strictly biological point of view. Genetically engineered SOD-null mutants in mice are normal except for a minor reduction in lifespan, inter se sterility and susceptibility to neuronal cell injury [8, 9]. In humans, mutations in SOD can lead to a familial form of the mid-life onset neurodegenerative disease, amyotrophic lateral sclerosis (FALS) [ 10, 11].
25 Reactive oxygen (RO) has also been identified as an important effector in aging and lifespan determination [12-14]. However, the specific cell types in which oxidative damage acts to limit lifespan of the whole organism have not been explicitly identified.
If these specific cell types can be identified, overexpression of SOD in theae cells may provide a means for delaying or inhibiting the aging process and thereby, increase lifespan.

SUMMARY OF THE INVENTION
The present invention involves the association between mutations in the oxygen radical metabolizing enzyme CuZn superoxide dismutase (SOD) and loss of motorneurons in the brain and spinal cord that occurs in the life-shortening paralytic disease, Familial Amyotrophic Lateral Sclerosis (FALS) [15], and chronic and unrepaired oxidative damage occurring specifically in motorneurons which is a critical causative factor in aging. Transgenic Drosophila were generated with expression of human SOD targeted to adult motorneurons. It is shown that overexpression of a single gene, SOD, primarily in a single cell type, the motorneurons, extends 1o normal lifespan by up to 40% and restores the lifespar~ of a short-lived SOD-null mutant.
Elevated resistance to oxidative stress suggests that the lifespan extension observed in these flies is mediated by enhanced RO metabolism. These results show that SOD activity in motorneurons is an important factor in aging and lifespan determination in Drosophila.
The present invention therefore provides a method of increasing reactive oxygen 15 metabolism in a cell, comprising inserting, in the cell, a gene encoding SOD and expressing the gene. In a further aspect of the present invention, the gene encoding SOD is inserted into a motorneuron cell. Further, the method of increasing reactive metabolim in a cell as defined above, prolongs the life of the cell.
In another aspect of the present invention, there is provided a method for increasing the 2o lifespan of a human or animal, comprising inserting a gene encoding SOD
into the cells of the human or animal, wherein the cells comprise motorneurons, and expressing the gene.
The present invention also provides a vector for the expression of a gene encoding SOD
in a cell comprising:
regulatory elements for expression of the gene; and 25 ~ the gene encoding SOD operatively associated wil;h the regulatory elements and capable of expression in the cell.
In a further aspect of the present invention, there is also provided a pharmaceutical composition comprising an effective amount of a SOD gene or a vector encoding a SOD gene and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS
The features of the present invention will become more apparent from the following description in which reference is made to the appended drawings i.n which:
Fig. 1. Illustrates GAL4-activated expression of HS in motorneurons. Whole mounts of adult brain and ventral ganglia hybridized in sit~c with a full length dioxygenin-labeled human SOD
(HS) cDNA. Tissues were examined from transgenic flies bearing one copy each of HS1 and D42-GAL4 (HS1/+; GAL4/+). (a) Transgenic HS expression was detected pimarily in the central l0 brain (Br), lateral margins adjacent to the lobula/lubula plate (arrowheads), and suboesophageal ganglia (S). No expression was detected in the optic lobes (OL) or retina (R).
(b) A schematic of the ventral ganglia depicting the location of four ganglionic regions:
prothoracic (Pro), mesothoracic (Meso), and combined metathoracic and abdominal ganglia (Meta-Ab). Peripheral nerves which act as landmarks are also shown, (ADMI'1, PDMN; L1 and L2). Four of the five 15 identifiable flight motorneurons (red circles) are ventrally located, the fifth is located dorsally. (c) The expression of the D42-GAL4 line was determined by immunofluorescence after crossing to flies containing a UAS-GFP transgene. Illustrated is the result of a z-series of confocal images through the ventral ganglia. The location of four of the large flight muscle motorneurons is indicated by an arrowhead. (d) Expression of HS can lbe detected within flight muscle 2o motorneurons 1-4 (*) as well as other motorneurons distributed at various locations within the ventral ganglia. Scale bar for (a) = 200 microns and for (b) = 100 microns.
Fig. 2. Illustrates the detection of transgenic HS protein. (A) Immunoblot analysis of adult extracts using an antibody to human SOD. The arrow indicates a 21 kD
immunoreactive protein 25 corresponding to human SOD (a) HS1/+; GAL4/+; (b) HS2/+;GAL4/+; (c) HS1/+;
+/+; (d) Authentic purified human SOD (0.025 g). The strains in lanes a-c were also homozygous for the Drosophila SOD+ genes. (B) Assay of SOD activity in nondenaturing polyacrylaminde gels (6)).
Residual Mn SOD activity is indicated by (*). (a) HS'?/HS2; +/+; (b) HS2/HS2;
GAL4/GAL4;
(c) HS 1 /HS 1; +/+; (d) HS 1 /HS 1; GAL4/GAL4; (e) +/+; GAL4/GAL4; (f7 wild type, Oregon R

strain. The strains in lanes a-a are homozygous for the SODx39 mutation that precludes formation of indigenous Drosophila SOD. SOD speci:flc activity (Units/mg protein) was also determined for each strain (30): (a) HS2/HS2; +/+: 0.10.8 ; (b) HS2/HS2;
GAL4/GAL4:
6.210.8 ; (c) HS 1 /HS 1; +/+: 0; (d) HS 1 /HS 1; GAL4/'GAL4: 7.52.6; (e) +/+;
GAL4/GAL4: 0.
Values represent the mean ~ SEM of 12 determinations (3 different extracts each assayed 4 times) after corrrection for residual Mn SOD activity. The "0" values are 1 SEM.
Fig. 3. Graphs depicting extension of normal adult lifespan by selective augmentation of HS in motorneurons. Adult SOD+ males (0-24 hrs old) bearing a single copy of HS 1 (A) or HS2 (B) to and either one or no copies of D42-GAL4 were maintained at 25°C in shell vials (10 flies per vial) containing standard cornmeal agar medium. The starting population size for each genotype was 250. Flies were transferred to fresh medium and scored for survivorship every two days. The mean (50% mortality) and maximum (90% mortality) lifespan for each genotype is as follows:
HS1/+;+/+ (mean =45.1 +/- 3.4; max. = 56.3 +/- 3.6); HS1/+; D42GAL4/+ (mean =
63.7 +/- 4.3;
max. = 73.2 +/- 3.4; HS2/+; +/+ (mean = 52.2 +/- 1.8; max. = 58.8 +/- 1.5);
HS2/+; D42GAL4/+
(mean = 60.6 +/- 2.2; max. = 71.0 +/- 2.7). The lifespan of the D42-GAL4/D42-GAL4; +/+
control is very similar to the HS/+; +/+ strains. In addition, expression of HS under the transcriptional control of other GAL4 drivers, including a heatshock-GAL4 driver which drives expression broadly at all stages of development and an elav-GAL4 driver which drives 2o expression at high levels in embryonic and larval neurons , did not extend lifespan (data not shown).
Fig. 4. Graph depicting restoration of adult lifespan i:n an SOD-null mutant by auxiliary expression of HS in motorneurons. (A) Adult males (0-48 hrs old) homozygous for the SOD-null mutation SODx39 and also bearing different combinations of HS and D42-GAL4 transgenes were maintained at 25°C in shell vials (10 flies per vial) containing standard cornmeal agar medium. The starting population size for each genotype was 50 flies. Flies were scored daily for survivorship and transferred to fresh vials every two clays. (B) Gene dosage effects on restoration of adult lifespan. Adult males (0-48 hrs old) homozygous for the SOD-null mutation SODx39 and also bearing one or two copies each of HS 1 and the D42-GAL4 activator were constructed and lifespan studies were conducted as in (A). The starting population sizes were 180, 33$ and 70 for the 0 dose, 1 dose and 2 dose genotypes, respectively. The zero dose control bears 2 copies of HS1 but no D42-GAL4 activator. The data presented are representative of at least two separate experiments.
Fig. 5. Bar graph representing the relationship between motorneuronal SOD and lifespan in Drosophila.
1o Fig. 6. Graphs depicting the resistance to oxidative stress of a SOD-null mutant conferred by expression of HS in motorneurons. (A) Resistance to paraquat. Adult males (0-48 hrs old) homozygous for the SOD-null mutation SODx39 and also bearing different combinations of UAS-HS and D42-GAL4 transgenes were maintained at 2$°C in shell vials containing filter pads saturated with aqueous paraquat and scored for survivorship after 24 hours.
Each point represents $0 flies ($ vials of 10 flies each). (B) Resisl;ance to ionizing radiation. Adult males (24-48 hrs old) homozygous for the SOD-null mutation SODx39 and also bearing different combinations of UAS-HS and D42-GAL4 transgenes were exposed to 100 lcRad -radiation (190 min at ~ $20 Rads/min in a cobalt60 source) and then maintained at 2$°C
in shell vials containing standard cornmeal agar medium and scored daily for survivorship.
The data are 2o representative of at least two separate experiments.
Fig. 7. Graph depicting the effect of the overexpression of catalase in motorneurons on lifespan of Drosphila. Adult CAT+/+ males (0-24 hr old) bearing a single copy of UAS-CAT (DC) and either one or no copies of the motorneuron driver D42-Gal4 (Gal4), were maintained at 2$oC on standard cornmeal agar medium at 10 flies/vial. The starting population size for each genotype was 100 animals.
Fig. 8. An immunoblot analysis depicting the detection of transgenic human SOD
protein.
Immunoblot analysis of adult extracts using an antibody to human SOD. (a) HS1/+; GAL4/+, (b) HS2/+; GAL4/+, (c) FS1/+; GAL4/+, (d) HS1/+; +/+, (e) Authentic purified human SOD (0.025 mg). The strains in lanes (a)-(c) were also homozygous for the Drosophila SOD+
genes.
Fig. 9. Bar graph depicting specific activity of transgenic human SOD in strains lacking Drosophila SOD. The (+) and (-) symbols indicate the presence (GAL4/GAL4) or the absence of the D42GAL4 enhancer element, respectively in that strain. The specific activities (Units/mg protein) for each strain are: (a) +/+; +/+: 0; (b) +/+; G.AL4/GAL4: 0; (c) hSODl/hSODl; +/+: 0;
(d) hSODI/hSODI; GAL4/GAL4: 7.512.6; (e) FS1/FS1; +/+: 1.50.04; (f) FS1/FS1;
GAL4/GAL4: 2.81.6; (g) FS2/FS2; +/+: 0; (h) FS2/hS2; GAL4/GAL4: 0.310.08.
Values 1o represent the mean of 12 determinations (3 different extracts each assayed 4 times). The "0"values are _1 SEM. The data for SOD1 has been published previously [16] and is presented here for comparison only. Note that the FS2 line is "leaky", showing substantial SOD activity in the absence of GAL4 activation.
Fig.10. Graph depicting survivorship of SOD+ flies expressing FALS SOD in motorneurons.
Adult SOD+ males (0-24 hr old) bearing a single copy of FS 1 (A) or FS2 (B) and either one or no copies of GAL4 were maintained at 25°C in shell vials (10 flies per vial) containing standard cornmeal agar medium. The starting population for each genotype was 250. Flies were transferred to fresh medium and scored for survivorship every two days. In this figure, the +
2o symbol indicates a non-transgene-bearing chromosome Fig. l 1. A bar graph depicting the resistance to paraquat of SOD+ flies expressing FALS SOD in motorneurons. Adult males (0-48 hr old) homozygous for the SOD-null mutatation and also bearing a single copy of HS 1 or FS 1 and either one (+) or no (-) copies of GAL4 were maintained at 25°C in shell vials containing filter pads saturated with 40 mM paraquat and scored for survivorship after 24 hours. Each point represents 100 flies (10 vials of 10 flies each). The data are representative of at least 2 separate experiments. Note that the relatively high resistance of the FS 1 (-) flies correlates with the "leaky" expression of FALS SOD in the absence of GAL4 activation.
Fig. 12. Bar graph depicting the eclosion efficiency of SOD- flies expressing FALS SOD in motorneurons. Crosses were set up between TM3-balanced SODxj9 heterozygotes that were also hemizygous for the appropriate combination of GAL4 and OAS-SOD transgenes. The percentage of successfully eclosed adult progeny that were homozygous for the SOD-null mutation SODx39 was calculated from the total progeny arising from these crosses. In addition the progeny were also homozygous for HS 1 or FS 1 transgenes or carried no OAS-SOD transgene l0 (SOD-), and carried either one (+) or no (-) copies of GAL4.
Fig. 13. Graph illustrating survivorship of SOD- flies expressing FALS SOD in motorneurons.
Adult males (0-48 hr old) homozygous for the SOD-null mutatation and also bearing one or no copies of FS 1 and either one or no copies of GAL4 were maintained at 25°C in shell vials (10 15 flies per vial) containing standard cornmeal agar medium. The starting population for each genotype was 250. Flies were tranferred to fresh medium and scored for survivorship every two days. In this figure, + indicates a nontransgene-bearing chromosome.
Fig. 14. Graph illustrating the survivorship of SOD+ :flies co-expressing FALS
SOD and human 2o wild type SOD in motorneurons. Adult SOD+ males ( 0-24 hr old) bearing single copies each of FS1 and HS1 and either one or no copies of GAL4 wf,re maintained at 25°C in shell vials (10 flies per vial) containing standard cornmeal agar medium. The starting population for each genotype was 250. Flies were tranferred to fresh medium and scored for survivorship every two days.
DETAILED DESCRIPTION OF THE INVENTION
There are at least 3 known forms of the CuZn superoxide dismutase (SOD) enzyme.
These forms have been designated SOD1, SOD2 and SOD3. SODI is found primarily in the cytoplasm. SOD2 is the form found mainly in mitochondria, whereas SOD3 is found mainly in the extracellular matrix. Unless otherwise stated, reference to SOD is meant to indicate SOD1.
Expression of a human SOD transgene (HS) in Drosophila motorneurons was achieved using the yeast GAL4/UAS system [16-18]. The D42-GAL4 activator used in these experiments is expressed broadly during embryogenesis but becomes restricted to motorneurons and interneurons within the larval nervous system and with the exception of a few unidentified neurons within the central brain, is restricted to motorneurons in the adult nervous system. The HS transgene consists of a human SOD cDNA coupled to a yeast UAS element within a Drosophila P transformation vector. Two independent UAS-HS transgenic lines, designated HS 1 and HS2, were used in these experiments. Because lifespan is strongly affected by variation in to genetic background, a series of genetic schemes was employed to introduce the D42-GAL4 and UAS-HS transgenes into uniform SOD+ or SOD genetic backgrounds and to construct expressing and non-expressing strains that were essentially co-isogenic.
In situ hybridization shows that expression of the HS transgene is limited to adult motorneurons including a set of five bilaterally symmetrical motorneurons which control flight muscles in Drosophila (Fig. 1). Similar results were obtained for whole mount preparations of Drosophila larvae (data not shown). To confirm that functional human SOD
proteins were expressed in these cells, whole fly extracts were analyzed by immunoblot and SOD activity assays (Fig. 2). Using an antibody to HS that does not: cross-react with Drosophila SOD, substantial HS protein was detected in flies arising from a cross between the D42-GAL4 and the 2o UAS-HS lines, while no HS protein was detected in the D42-GAL4 line nor in either of the 2 UAS-HS lines. Assay of SOD activity in GAL4-UAS:HS transgenic flies that are also homozygous for a SOD-null mutation demonstrates that the transgenic HS is enzymatically active.
To determine the consequences of SOD overexpression in motorneurons on normal longevity, the D42-GAL4 and UAS-HS transgenes were genetically introduced into flies with a normal SOD+ genetic background. Previous studies in which SOD levels had been increased broadly throughout many tissues showed little to no effect on adult lifespan in Drosophila [ 19-21] unless combined with a similar increase in catalase [22]. In contrast, it was found that if SOD overexpression is targeted selectively to motorneurons it causes a dramatic extension of lifespan (Fig. 3). Transgenic HS 1 flies overexpressing SOD in motorneurons exhibit mean and maximum adult lifespans up to 40% longer than non-expressing isogenic controls. The most striking feature of the postponed mortality in these flies is the extension of the premortality plateau phase of the life curve (<5% mortality) from approximately 27 days to 50 days. That is, selectively enhanced expression of SOD in motorneurons nearly doubles the time before the onset of significant mortality. The HS2 strain, which exhibits SOD activities without and with GAL4 activation of 1.3% and 83%, respectively, of the level of activity in activated strain HS1, confirms the relationship between the level of motorneuron-enhanced SOD
activity, postponed senescence and lifespan extension. These results demonstrate that enhancing SOD activity in motorneurons can markedly postpone the age-dependE;nt onset of senescent mortality in Drosophila. It may be concluded from these results that RO metabolism specifically in motorneurons is a critical factor in senescence and life;span determination in Drosophila.
One of the characteristics of SOD-null mutants of Drosophila is the foreshortening of the adult lifespan by 85-95%. Complete rescue of normal lifespan in SOD-null mutants is afforded by a genomic Drosophila SOD transgene that expresses SOD in the normal pattern throughout the body [23]. The results, above, predict that lifespm of a SOD-null mutant would also be substantially restored if SOD were selectively expressed just in motorneurons.
To test this prediction, the D42-GAL4 and HS transgenes were introduced into flies with a SOD-null mutant genetic background and lifespans were determined. T'he SOD mutation used in these experiments, SODx39 is an internal deletion that precludes synthesis of SOD
protein [24]. As can been seen (Fig.4), selective expression of SOD in motorneurons restores the lifespan of SOD-null mutant flies in a clearly dose-dependent manner. Flies bearing one or two copies each of the D42-GAL4 and UAS-HS transgenes exhibit mean adult lifespans of approximately 10 and 36 days, respectively, compared to the 3 day mean lifespan of the isogenic SOD-null mutant control.
This represents a restoration of the lifespan from 5% (exhibited by the SOD-null mutant) to greater than 60% of the isogenic SOD+ control. The :full range of lifespans produced in this study by controlling the expression of SOD in motorn.eurons of flies with SOD-null and SOD+
genetic backgrounds is summarized in Figure 5.
That restoration of lifespan is not complete shows that there is a requirement for SOD and RO metabolism in other tissues. However, we have examined the effects of enhanced SOD
expression in several other tissues during development and not observed any significant ability to enhance lifespan (data not shown). The impressive rescue achieved by overexpression of SOD in motorneurons implies that motorneuron dysfunction arising from the lack of SOD
is the principal cause of the reduced lifespan of SOD-null mutants.
To determine if the mechanism of lifespan extension involves the catalytic activity of SOD and enhanced superoxide metabolism in motorneurons, flies bearing combinations of UAS-HS l and D42-GAL4 transgenes were oxidatively stressed by exposure to the RO-generating agents paraquat and ionizing radiation (Figure 6). Expression of HS in motorneurons provided 10 significant resistance to both challenges. While not wishing to be limited by theory, the results of the study strengthens the view that the mechanism underlying extended lifespan in these flies involves elevated RO metabolism in motorneurons.
To determine if the observed extension of lifespan in our transgenic HS lines could be attributed to lower metabolic rates we measured respiration rates in flies which expressed HS in motorneurons as compared to controls. The results demonstrate that activation of the HS
transgene in motorneurons does not depress metabolic: rate, effectively excluding this as a possible mechanism for the lifespan extension seen in these experiments.
The results of this study show an important rei:inement of the free radical (oxidative damage) hypothesis of aging [12, 13], namely, that lifespan is determined by RO metabolism in a 2o small number of critical cell types that includes motorneurons.
In further experiments it has been shown that expression of a human FALS SOD
gene, which specifies a mutant SOD enzyme with lower enzymatic activity, in Drosophila motorneurons:
a) does not confer neurodegeneration, paralysis and premature death as it does in humans;
b) extends the median (but not the maximum) lifespan of both SOD' and SOD-flies;
c) enhances the resistance of both SOD' and SOD- flies to oxidative stress.
Compared to the previous results with the human SOD+ gene, the effects of the mutant FALS SOD gene on lifespan extension are modest. For example, the FALS SOD gene gives only 10% extension of lifespan compared to 40% with the SOD+ transgene. This result is 3o attributed to the reduced enzymatic activity of FALS SOD compared to normal human SOD.

Overall, these results give unambiguous and independent confirmation of the results of experiments with the human SOD' gene. Moreover, they confirm the result that the effects of motorneuron-targeted expression of SOD on both lifespan and resistance to oxidative stress are quantitatively correlated to the level of SOD enzymatic activity conferred by the SOD transgene.
That is, higher levels of total SOD enzymatic activity, whether arising by higher levels of transgene expression, or by SOD alleles that specify SOD with higher intrinsic specific activity, or both, have correspondingly greater effects on lifespan extension and resistance to oxidative stress.
Further experiments have shown that the targeted expression of a gene other than SOD in motorneurons has no effect on lifespan. Specifically, overexpression of the catalase gene in motorneurons does not extend lifespan (Figure 7). Catalase is an enzyme that is also involved in reactive oxygen metabolism, but catalysing a different: reaction than SOD.
Importantly, this result demonstrates that the results obtained through the targeted expression of SOD to motorneurons can be attributed specifically to the use of SOD, and are not obtained with another gene or enzyme, even one otherwise involved in reactive oxygen metabolism.
This result does not rule out the possibility that the other forms of SOI), namely SOD2 and SOD3, if overexpressed in the motorneurons, would result in increased lifespans.
The gradual diminution of motor function is one of the hallmarks of aging in animals and has significant ramifications in gerontology. Moreover, the sensitivity of motorneurons to 2o oxidative impairment is well documented in both vertebrates and invertebrates. Our ability to substantially postpone senescent mortality and to alleviate mutant symptoms arising from impaired RO metabolism by antioxidant intervention in motorneurons in Drosophila suggests a possible strategy for reducing the morbidity of normal senesence in other animals.
This invention relates to a method of reducing reactive oxygen (RO) damage by expressing SOD genes in cells. The cells transformed with the SOD gene are preferably nervous system cells. In a more preferred embodiment, the cells are motorneurons.
In embodiments of the invention, the method of reducing reactive oxygen damage by expressing SOD genes in cells, in particular motorneuron cells, may be applied to humans or any animal, including companion animals and livestock such as cows, pigs, horses, chickens and the like.

The present invention also relates to a method of increasing the lifespan of a human or animal by expressing SOD in the cells of a human or animal, wherein the cells comprise motorneurons. The term "increasing lifespan" as used herein means to increase the useful lifespan of a human or animal, that is, to delay the onset of normal age-related deterioration of cellular function, and thereby increasing the more productive period of a human or animal's life.
Normal age-related deterioration of cellular function rnay be evidenced at several levels. At a cellular level, normal age related deterioration may be manifested by (among other factors) loss of mitochondria) function and reduced energy generation, loss of membrane plasticity and cellular integrity, and increased susceptibility to cell death triggered by oxidative stress. At the to organism level, normal age related deterioration of cellular function may be manifested by (among other factors) loss or reduction of neural functions including memory, cognitive ability, attention span, reaction to stimuli and neuroendocrine activity (hormone secretion), loss or reduction of neuromuscular function including strengl:h, endurance, reflex reaction and locomotor activity, and loss or reduction of tissue plasticity including increased brittleness of bones and wrinkling skin.
In embodiments of the invention, the method of increasing the lifespan of a human or animal by expressing SOD in the cells of the animal wherein the cells comprise motorneurons, may be applied to humans or any animal, including companion animals and livestock such as cows, pigs, horses, chickens and the like.
2o The invention includes nucleotide modifications of the gene sequences disclosed in this application (or fragments thereof) that are capable of producing SOD proteins.
The SOD gene from any organism may be used to increase RO metabolism. In a preferred embodiment, the gene is the SOD gene from a mammal. In the most preferred embodiment, the gene is the human SOD gene. The SOD gene sequence may be modified using techniques known in the art.
Modifications include substitution, insertion or deletion of nucleotides or altering the relative positions or order of nucleotides. The invention includes DNA which has a sequence with sufficient identity to a nucleotide sequence described in this application to hybridize under stringent hybridization conditions (hybridization techniques are well known in the art). The SOD genes that may be used to increase RO metabolism of the invention also include genes (or a 3o fragment thereof) with nucleotide sequences having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96'% identity, at least 97% identity, at least 98% identity or, most preferred, at least 99% identity to the SOD gene sequences of the invention. Identity is calculated according to methods known in the art. For example, if a nuceotide sequence (called "Sequence A") has 90% identity to a portion of the SOD gene, then Sequence A will be identical to the referenced portion of the SOD gene except that Sequence A
may include up to 10 point mutations (such as deletions or substitutions with other nucleotides) per each 100 nucleotides of the referenced portion of t:he SOD gene. Other SOD
genes may also be used to increase RO metabolism, including SOD2 and SOD3.
The DNA sequences of the invention (regulatory element sequences and therapeutic gene l0 sequences) may be obtained from a cDNA library, for example using expressed sequence tag analysis. The nucleotide molecules can also be obtained from other sources known in the art such as genomic DNA libraries or synthesis.
The genes are preferably expressed by vectors containing DNA regulatory elements that direct the expression of the genes for use in research, protein production and gene therapy in cells and tissues (preferably animal cells and tissues). Viral vectors may be used, for example, adenovirus vectors. For example, in addition to the expression vector described in the examples above, the promoter for neuronal enolase gene or the promoter that specifies neurofilaments or VAMPl (synaptobrevin) may be used to express genes. Other regulatory elements from other mammals, yeast, bacteria, viruses, birds or insects ma;y also be used to express the genes. The 2o specific regulatory elements chosen for a particular vector may vary depending on factors such as the level of activity of the cassette desired or the characteristics of the gene to be expressed. One skilled in the art can modify the sequences of the regulatory elements and the gene to be expressed using techniques disclosed in this application and known in the art.
The SOD gene may be expressed to prevent RO damage in vivo or in vitro. Cells transformed in vitro can be used as a research tool. The gene is also useful for gene therapy by transforming cells in vivo to express a therapeutic protein that increases RO
metabolism. Gene therapy may be used to treat diseases, disorders and abnormal physical states such Amylotrophic Lateral Sclerosis, Huntington's disease, Parkinson's disease and Alzheimer's disease and any other disease, disorder or abnormal physical state of the motorneurons induced by reactive oxygen. In a preferred embodiment of the present invention gene therapy may be used to delay the onset of normal aging in an animal and thereby, increase the lifespan of that animal.
The SOD protein is one therapeutic protein which may be expressed in vivo or in vitro to increase RO metabolism. Other SOD proteins may also be used to increase RO
metabolism.
These include the proteins produced by SOD2, SOD3 or fragments thereof.
Changes in the nucleotide sequence which result in production of a chemically equivalent (for example, as a result of redudancy of the genetic code) or chemically similar amino acid (for example where sequence similarity is present), may also be used as therapeutic proteins with the expression cassettes of the invention.
i0 Pharmaceutical compositions used to treat animals to increase their lifespan or treat disease states include a SOD gene or SOD protein and an acceptable vehicle or excipient (Remington's Pharmaceutical Sciences 18"' ed, 1990, Mack Publishing Company and subsequent editions). Vehicles include saline and DSW (5% dextrose and water). Excipients include additives such as a buffer, solubilizer, suspending agent, emulsifying agent, viscosity controlling agent, flavor, lactose filler, antioxidant, preservative or dye. There are preferred excipients for stabilizing peptides for parenteral and other administration. The excipients include serum albumin, glutamic or aspartic acid, phospholipids and fatty acids. The gene or protein may be formulated in solid or semisolid form, for example pills, tablets, creams, ointments, powders, emulsions, gelatin capsules, capsules, suppositories, gels or membranes.
Routes of administration include oral, topical, rectal, parenteral (injectable), local, inhalant and epidural administration. The compositions of the invention may also be conjugated to transport molecules to facilitate transport of the molecules. The; methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients are known in the art.
The pharmaceutical compositions can be administered to humans or animals.
Dosages to be administered depend on individual patient condition, indication of the drug, physical and chemical stability of the drug, toxicity, the desired effect and on the chosen route of administration (Robert Rakel, ed., Conn's Current Therapy, 1995, W.B. Saunders Company, USA). The pharmaceutical compositions are used to increase lifespan or treat diseases, disorders 3o and abnormal physical states described above in this application.
Preferably the pharmaceutical 1$
compositions are used to increase the lifespan of an animal.
The present invention has been described in teams of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. All such modifications are intended to be included within the scope of the appended claims.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was 1o specifically and individually indicated to be incorporated by reference in its entirety.
EXPERIMENTAL EXAMPLES
EXPERIMENT 1: EXPRESSION OF HUMAN SOD IN MOTORNEURONS OF
DROSPHILA
Transgenic strains. The UAS-HS transgene was constructed by inserting an HS
cDNA in the polycloning site behind the UAS element of the P expression vector, pUAST, that contains a miniwhite+ reporter gene. Transformants were made using whitel recipients and standard P
2o transformation methodology [25]. The D42-GAL4 strain carries a 3rd chromosome GAL4 enhancer trap that selectively expresses the GAL4 traliscriptional activator in adult motorneurons [18].
Genetic constructions. To generate flies with D42-C?AL4-activated expression of 2nd chromosome P(w+)UAS-HS transgenes and appropriate non-activated controls in an isogenic Drosophila SOD+ background, homozygous wl;P(w ~)UAS-HS; + females were mated in parallel to wl recipient strain males and to homozygous wl;+;p(w+)D42-GAL4 males.
wl;P(w+)UAS-HS/+;P(w+)D42-GAL4/+ males, carrying one copy of each of the transgenes and therefore showing D42-activated SOD expression, could then be compared directly to wl;P(w+)UAS-HS/+;+/+ males lacking expression.
To generate flies homozygous for both a UAS~-HS transgene and the D42-GAL4 activator in a SOD-null mutant background, it was necessary to construct a recombinant chromosome carrying both the P(w+)D42-GAL4 insert and the SODx39 mutation because both of these genetic elements reside on chromosome III. The D42R20 (D42-GAL4 , SOD'c39) and (D42-GAL4+, SODx39) third chromosomes were constructed in parallel through a crossing scheme involving recombination between chromosomes carrying the P(w+) D42-GAL4 insert and the SOD'x39 mutation, the latter chromosome marked with the recessive eye colour marker, red. Based on the relative frequencies of each progeny class, the D42R20 and l0 chromosomes could be inferred to differ by at most the ~26 cM segment of chromosome III
between the SOD and red loci, or by as little as the P(,w+)D42-GAL4 insert itself. A crossing scheme employing a wl stock carrying both 2nd and ~;rd chromosome balancers was devised for construction of GAL4-UAS doubly balanced stocks to minimize variation in genetic background between stocks. Based on this crossing scheme, each of the four resulting GAL4-UAS stocks will carry wl chromosomes from identical sources, each of which derive ultimately from the X
chromosome of the original wl recipient strain. The second chromosomes of each stock, having been balanced throughout, will also directly derive from the wl recipient strain, and will differ only in the position of the P(w+)UAS-HS inserts. Apart from the presence or absence of P(w+)D42-GAL4, the recombinant third chromosomes of each stock will also be very similar, as described above. Thus, the experimental GAL4-UAS-HS stocks and their controls can be considered as virtually isogenic for most of the genorr~e, with minimal differences in the genetic background between strains. This allows us to attribute phenotypic characteristics specifically to the GAL4-activated HS expression without concern for phenotypic effects arising from other differences in genetic background.
To obtain expression of a single copy of a UAS-HS transgene activated by a single copy of the D42-GAL4 activator in a SOD-null mutant background, doubly balanced stocks carrying a UAS-HS transgene on the second chromosome and the SOD-null allele, SODx39, on the third chromosome were constructed by standard genetic techniques. Virgin females of the genotype wl lwl ;UAS-HSlUAS-HS; SODx39/TM3 were collected and mated in parallel to w1;D42R40(D42-GAL4, SODx39)/TM3 males and to w1;D42R20(D42-GAL4 , SODx39)lTM3 males. Male progeny of the genotype w; UAS-HS/+;D42R40/SODx39, carrying single copies of both the P(w+) UAS-HS transgene and the P(w+)D42-GAL4 activator, were collected for direct comparison to male sibs of the genotype wl;UAS-HS,~+;D42R20/SODx39, carrying one copy of the P(w+)UAS-HS transgene but no P(w+)D42-GAL4 activator, in a SOD-null mutant genetic background that is co-isogenic for most of the genome. It is important to note here that the SODx39 allele used to make the SOD-null background contains a 365 by deletion that includes the both the transcription and translation start sites and therefore makes neither a transcript nor a protein product [24]. Thus, the only SOD protein in these flies is that specified by the UAS-HS
transgene. This prevents any possible interference by indigenous Drosophila SOD subunits in the formation of homodimeric HS.
Motorneuron specificity of D42-GAL4. Expression of the D42-GAL4 activator was determined by crossing the D42-GAL4 line to a UAS-GFP (green fluorescent protein) line followed by fluorescence microscopy, or to a UAS-lacZ line followed by immunocytochemistry using an anti-13-galactosidase antibody. The pattern of D42-GAL4 in embryos is described elsewhere [18].
In larvae, D42-GAL4 is expressed in motorneurons, interneurons and some peripheral glial cells.
2o Low levels of expression were also detected in the fat body. In the adult, D42-GAL4 expression is restricted to a small number of cells within the central brain and to motorneurons within the ventral ganglia.
SOD assay. SOD activity was assayed qualitatively following electrophoresis in nondenaturing polyacrylamide gels [26]. For quantitative assay of SOD activity, extracts were made by homogenizing 20 adult males (24 hr old and previously frozen at -80°C) in 200 ml of 0.05 M
sodium phosphate pH 7.4, 0.1 mM EDTA. After centrifugation at 13,000 g for 5 min at 4°C, the supernatants were partially deproteinized by treatment with chloroform/ethanol [27] and assayed for CuZn SOD activity by the 6-hydroxydopamine autoxidation method [28].
Proteins were determined using the Bio-Rad Protein Assay Kit.
Lifespan Determination. For statistical analysis, the mean and maximum (90%) lifespan of each strain was calculated from the time (in days) at which survival reached 50% and 10% of the starting population in each of the 25 cohorts of each strain. The means and variances of these estimates were calculated, and used to establish a 99°/~ confidence interval for mean and maximum lifespan values. The values calculated are ;~s follows:
HS1/+; +/+............mean 45.1 +/- 3.4.........max 56.3 +/- 3.6 HS 1/+;D42/+..........mean 63.7 +/- 4.3.........max 73.2 +/- 3.4 io HS2/+;+/+.............mean 52.2 +/- 1.8.........max 58.8 +/- 1.5 HS2/+;D42/+..........mean 60.6 +/- 2.2.........max 71.0 +/- 2.7 Oxidative Stress. Adult flies were challenged with p~~raquat and ionizing radiation as described in Parkes et al. [23].
Oxygen Consumption. Oxygen consumption was measured with a Gilson Single-Valve Differential Respirometer using standard methods (Umbreit, 1964). Twenty-five flies were weighed, placed in the respirometer and left undisturbed for 2 hours at 25°C. The rate of change of volume was expressed as uL of dry 02 consumed per mg of wet weight per hour at standard temperature and pressure (STP). Rates were examined for differences with 1-way ANOVAs, and 2o means were compared using the SNK test.
EXPERIMENT 2: EXPRESSION OF HUMAN FALS ALLELE IN THE
MOTORNEURONS OF DROSPHILA
Human FALS Allele The SOD allele used in this study was originally identified in a large Italian FALS
pedigree characterized by a mean age at onset of symptoms of 47 years with a rapid progression leading to death in less than one year [29]. The allele involves a GGC -> AGC
transition mutation in codon 41 within the 2nd exon that leads to a G41 S missense substitution. The specific activity of SOD from heterozygous patients in this pedigree is reportedly reduced by 27% compared to unaffected siblings [30].
Transgenic strains UAS-SOD transgenes were constructed by inserting a FALSSOD cDNA into the polycloning site behind the UAS element of the P expression vector, pUAST, that contains a miniwhite+ reporter gene. UAS-SOD transformants were made using white' recipients and standard P transformation methodology [25]. Two independent transgenic lines, FS1 and FS2, were generated; however most of the data reported here was obtained from the FS 1 line which gave the most robust expression. The D42-GAL4 strain carries a GAL4 enhancer trap P element to vector that expresses the GAL4 transcription factor in adult motorneurons [18]. Expression of FALS SOD was obtained by crossing the D42GAL4 and UAS-SOD lines. The transgenic strain (designated HS1) expressing wild type human SOD was obtained as described in Experiment 1.
Genetic constructions Flies with D42GAL4-activated expression of UAS-SOD transgenes and appropriate non-activated controls were constructed in co-isogenic SO.D+ and SOD- backgrounds as described in Experiment 1. The SODx39 allele used to make the SOD-null background contains a 365 by deletion that includes both the transcription and translation start sites and therefore makes neither a transcript nor a protein product [24]. Thus, the only SOD protein in these flies is specified by the UAS-SOD transgene and potential interaction between transgenic human SOD
subunits and indigenous Drosophila SOD subunits is precluded.
SOD assay Transgenic human SOD protein was determined using an anti-human SOD antibody to probe Western blots of extracts of SOD-null mutants as described above. SOD
enzymatic activity was assayed qualitatively following electrophoresis in nondenaturing polyacrylamide gels [31, 26]. For quantitative assay of SOD activity, extracts were made by homogenizing 20 adult males (0-24 hr old and previously frozen at -80°C) in 200 ml of 0.05 M sodium phosphate pH 7.4, 0.1 mM EDTA. After centrifugation at 13,000 g for 5 min at 4°C, the supernatants were partially deproteinized by treatment with chloroform/c;thanol [27] and assayed for CuZn SOD
activity by the 6-hydroxydopamine autoxidation method [28]. Proteins were determined using 3o the Bio-Rad Protein Assay Kit.

Oxidative stress Adult flies were challenged with paraquat as described in Experiment 1.
Briefly, adult males (0-24 hr old) were collected, allowed to recover from ether anesthesia for 12 hrs (on standard cornmeal agar medium at 25°C, and then maintained at 25°C in the dark in shell vials 5 containing filter pads saturated with 250 ml of freshly prepared 40 mM
aqueous paraquat and scored for survivorship after 24 hours.
Lifespan determination Adult lifespan was determined as described in Experiment 1. Briefly, adult males (0-24 hrs old) were maintained at 25°C in shell vials (10 flies per vial) containing standard cornmeal to agar medium. The starting population size for each genotype was 250. Flies were transferred to fresh medium and scored for survivorship every two days Targeted expression of G41S FALS SOD in adult rnotor neurons The yeast GAL4/ UAS transcriptional regulatory system was used to direct expression of human wild type or FALS mutant SOD (G41 S) in motorneurons of adult Drosophila. The D42 15 GAL4 enhancer trap line, which expresses GAL4 in adult motorneurons, was used to drive expression of UAS-linked human SOD genes and is described elsewhere [18].
Expression of the human wild type and FALS SOD protein was measurE;d in extracts of whole flies by Western blot analysis after crossing the UAS-SOD transgenes into the D42GAL4 line.
Comparable levels of human wild type and FALS SOD protein were detected (Figure 8) That this protein is 20 enzymatically active is shown by activity measurements of SOD in extracts of flies expressing the UAS-SOD transgene in a SOD-null genetic background (Figure 9). Note that the UAS-FS 1 transgene is somewhat leaky, but still responds to GAL4 activation. However, despite comparable levels of SOD protein, the FALS SOD protein shows significantly reduced activity, demonstrating that the G41S human FALS SOD protein behaves as a loss-of function or hypermorphic mutation in Drosophila. The inference from earlier experiments on mutant carriers [18] that the enzymatic activity of mutant G41S homodimeric SOD is severely reduced is now clearly confirmed by these results.
Expression of FALS SOD in motorneurons is not deleterious and extends adult lifespan The overt manifestation of FALS in humans and in FALS-SOD transgenic mice is progressive paralysis leading to premature death. We have therefore used lifespan as a primary indicator of motorneuron dysfunction that might arise from FALS SOD expression in motorneurons. Figure 10 shows the survivorship data for wild type flies expressing FALS SOD
in motorneurons. Expression of FALS SOD in motorneurons clearly has no adverse impact on lifespan. In fact, it confers a marked enhancement of :lifespan that is proportional to the level of measureable SOD enzymatic activity.
Oxidative stress does not potentiate deleterious effects of FALS SOD
Although expression of FALS SOD does not elicit a deleterious phenotype in otherwise normal flies under normoxic conditions, it was reasoned that FALS SOD might become deleterious under conditions of oxidative stress . Because substantial evidence suggests that l0 FALS SOD can act as a pro-oxidant [32-33], it was question whether FALS SOD
would enhance the sensitivity of otherwise normal flies to oxidative stress imposed by paraquat, a redox cycling agent to which Drosophila mutants exhibit exquisite SOD-dependent hypersensitivity. Using a standard paraquat toxicity assay [26] (Figure 11), it call be clearly seen that expression of FALS
SOD in motorneurons of SOD+ flies in fact reduces, rather than increases, sensitivity to paraquat.
Thus, the oxidative stress conferred by paraquat does not elicit a latent or potential deleterious effect of FALS SOD in motorneurons.
Drosophila SOD does not suppress potential deleterious effects of FALS SOD
In humans and mice, FALS mutations have been shown to have dominant gain-of function effects on motorneuron survival. In contrast, the experiments above clearly show that a human FALS SOD transgene has no comparable deleterious effects when expressed in motorneurons of Drosophila. However, it remains possible that in Drosophila, potential deleterious effects of FALS SOD are masked by the presence of Drosophila wild type SOD. To investigate this, the D42GAL4 and UAS-SOD transgenes were crossed into flies with an isogenic SOD-null genetic background and measured the effect on lifespan. Among other traits, SOD-null mutants normally exhibit reduced eclosion efficiency (difficulty completing metamorphosis) and severely reduced adult lifespan (~10-20% of nornial). If wild type Drosophila SOD
suppresses the deleterious effects of FALS SOD, it is predicted that one or both of these phenotypes would be exaggerated in SOD-null flies e:~pressing FALS SOD in motorneurons. As is shown in Figures 12 and 13, this is not the case. On the contrary, expression of FALS SOD

improves both lifespan and eclosion efficiency in a manner consistent with the level of enzymatic activity associated with G41 S SOD.
Co-expression of human wildtype SOD does not potentiate deleterious effects of FALS SOD
in motorneurons The dominant expression of FALS in human h.eterozygotes suggests that neurodegeneration may involve a mechanism that includes an interaction between mutant and wild type SOD. While Drosophila and human SOD are structurally very similar, they are not identical [24], so to investigate the possibility that development of FALS-type pathology in Drosophila requires interactions between FALS SOD and wild type human SOD that do not l0 occur with wild type Drosophila SOD, flies co-expressing both human FALS
SOD and human wild type SOD in motonneurons were generated and the effect on lifespan measured. Figure 14 shows that co-expression of both wild type and mutant human SOD in motorneurons is not detrimental to lifespan, and in fact confers a marked extension of lifespan as predicted by the aggregate increase in SOD activity in motorneurons ['.34].
EXPERIMENT 3: EXPRESSION OF CATALAS:E IN MOTORNEURONS OF
DROSPHILA
In this experiment, a gene encoding the enzyme catalase, which catalyzes the conversion of hydrogen peroxide, the product of the reduction of superoxide by SOD, to water, was expressed in motorneurons of normal CAT+ flies with no consequent effect on lifespan. This experiment was done to test the previous conclusion that the lifespan extending effect of overexpressing SOD in motorneurons depended upon the specific biochemical properties of SOD, and not on some general effect of the overexpression of any gene nor to a nonspecific effect of enhanced reactivie oxygen metabolism in general. Technically, this experiment was carried out precisely as described in Experiment 1 for SOD using the Gal4/UAS
regulatory system. A UAS-CAT transgene was made by fusing a Drosophila CAT cDNA to the yeast UAS
regulatory sequences and transformants bearing the transgene were generated.
UAS-CAT flies were then crossed to flies carrying the D42Ga14 driver transgene which were used and described in Experiment 1. The offspring, carrying both UAS-(:AT and D42Ga14 overexpressed transgenic CAT in their motorneurons but did not live any longer than the controls.
Analysis of transgene expression and lifespan were carried out exactly as described in Experiment 1.

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Claims (15)

1. A method of increasing reactive oxygen metabolism in a cell, comprising inserting in the cell a gene that encodes SOD and expressing the gene.

2. The method of claim 1, wherein the cell is a motorneuron.

3. The method of claim 1, wherein increasing reactive oxygen metabolism prolongs the life of the cell.

4. The method of claim 1, wherein the gene comprises a gene selected from the group consisting of a gene having at least 70% sequence identity with SOD and encoding a protein having SOD activity, and a gene encoding a protein having SOD activity.

5. The method of claim 1, wherein the gene comprises a gene selected form the group consisting of SOD, SOD2, SOD3 and fragments of the aforementioned genes.

6. A method for increasing the lifespan of a human or an animal, comprising inserting a gene encoding SOD into the cells of the human or animal, wherein the cells comprise motorneurons, and expressing the gene.

7. The method of claim 6, wherein the animal is selected from the group consisting of companion animals and livestock.

8. A method of for increasing the lifespan of a human or an animal comprising:
~ administering to the human or animal an amount of a SOD gene within an expression cassette so that the expression cassette is inserted in the human or animal's cells;
~ expressing the SOD gene to produce the SOD protein so that the SOD protein increases reactive oxygen metabolism.

9. A vector for the expression of a SOD gene in a cell, comprising ~ regulatory elements for expression of the SOD gene, and ~ the SOD gene operatively associated with the regulatory elements and capable of expression in the cell.

10. The vector of claim 9, wherein the cell comprises a motorneuron.

11. The vector of claim 9, wherein the gene comprises a human SOD gene.

12. The vector of claim 9, wherein the gene comprises a gene selected from the group consisting of a gene having at least 70% sequence identity with SOD and encoding a protein having SOD activity, and a gene encoding a protein having SOD activity.

13. The vector of claim 9, wherein the gene comprises a gene selected form the group consisting of SOD, SOD2, SOD3 and fragments of the aforementioned genes.

14. A pharmaceutical composition comprising a therapeutically effective amount of the SOD
gene and a pharmaceutically acceptable carrier.

15. A pharmaceutical composition comprising a therapeutically effective amount of the vector of any of claim 10 and a pharmaceutically acceptable; carrier.