HK1029532B - Erythropoietin in the treatment of domestic and livestock animals for anemia - Google Patents

Description

Erythropoietin for treating anemia in domestic and farm animals

The present invention relates to gene therapy methods for use in domestic and farm animals. In particular, the invention relates to methods of treating anemia in animals by administering expression vectors encoding erythropoietin, and pharmaceutical compositions containing said expression vectors.

Anemia arising from chronic renal failure, administration of chemotherapeutic drugs or surgery is one of the major causes of death and disability in domestic and farm animals. In addition to blood transfusions, the only method currently available for treating anemia in dogs, cats or pigs is the administration of recombinant human Erythropoietin (EPO). Although this treatment has the potential to improve the quality of life of the animal, it has two major drawbacks. First, such treatments are expensive and inconvenient (animals typically must be injected three times a week for life). Second, treated animals often produce antibodies to human proteins, reducing their efficacy, and may cross-react with the animal's own EPO, exacerbating anemia (Cowgill et al, J.Am.vet.Med.Assoc.) 212: 521-. Recent acquisition of dog-, cat-and pig-type EPO (Wen et al, Blood 82: 1507-. However, treatment is likely to be expensive and require frequent repetition. A method of EPO administration that circumvents these problems would be a significant advance in the veterinary art.

The present invention is based on the discovery that anemia in dogs, cats, and pigs can be alleviated by administration of an expression vector encoding EPO. This vector is taken up by cells in vivo and produces sufficient hormone for a prolonged period of time (3-4 months) to significantly increase the level of blood cell counts in the animal. By using vectors encoding EPO of the type found in treated animals (dog EPO to dog, cat EPO to cat, and pig EPO to pig), immunological problems caused by the administration of human EPO to these animals can be avoided.

In one aspect, the invention relates to a method of treating anemia in dogs by administering an expression vector containing a unique EPO sequence element consisting essentially of a sequence encoding dog erythropoietin. The term "consisting essentially of …" refers to a nucleic acid sequence containing the correct amino acid sequence encoding canine EPO, as well as nucleic acid sequences encoding proteins with insubstantial amino acid differences as evidenced by the retention of the essential functional and immunological properties of canine hormones. It is particularly important in this respect that the encoded EPO does not cause the production of anti-EPO antibodies in the treated animals. The EPO sequence element encodes the amino acid sequence of the mature EPO protein, which for canine EPO, has 166 residues. At its N-terminus, there is typically a signal sequence (preferably a homologous signal sequence). The term "homologous signal sequence" refers to a signal sequence normally associated with the encoded EPO, e.g., the canine EPO signal sequence will preferably be used for the canine EPO structural sequence. The complete amino acid sequence of the dog hormone is shown in FIG. 1(SEQ ID NO: 1). The first 26 amino acids (underlined in the figure) are the dog signal sequence, while the remaining amino acids are the structural sequence of mature dog EPO.

Expression vectors for use in treating animals should preferably contain promoter elements operably linked to EPO sequence elements. The term "operably linked" means that the two sequence elements (i.e., the promoter and the EPO structural sequence) are linked in such a way that transcription proceeds under the control of the promoter and EPO protein is properly synthesized. The expression vector should be administered to the animal receiving treatment in an amount sufficient to cause a statistically significant increase in its blood cell count.

Many different drugs have been described for enhancing cellular uptake of administered nucleic acids in vivo. While these agents are suitable for the present invention, the expression vector is preferably administered in admixture with a cationic lipid, as part of a liposome, or in the form of a non-transfection-facilitating agent. The expression vector should generally be administered at a concentration of 0.01-10mg/ml, with a preferred concentration being about 0.1 mg/ml. The preferred method of delivery is by intramuscular injection, but other methods, such as by a "gene gun" method, may also be used.

In another aspect of the invention, the methods discussed above for treating anemia can be applied to cats and pigs by replacing the canine EPO sequence element in the expression vector with a feline EPO sequence element or a porcine sequence element. Thus, an expression vector containing a unique EPO sequence element consisting essentially of nucleotides encoding feline EPO operably linked to a promoter element can be administered to a cat. For swine, the unique EPO sequence elements in the expression vector will consist essentially of the nucleotide encoding the porcine EPO. Like dogs, the encoded EPO should generally have a signal sequence at the N-terminus and the treated animals should be administered an amount of expression vector sufficient to cause a statistically significant increase in their blood cell count. The expression vector to be administered is preferably mixed with the cationic lipid, incorporated as part of the liposome, or administered in a form free of any transfection-facilitating drug. The sequence of feline EPO is shown in FIG. 2(SEQ ID NO: 2). The underlined amino acids in the figure are the feline signal sequence. The structural sequence of porcine EPO is shown in FIG. 3(SEQ ID NO: 3).

The invention also includes pharmaceutical compositions for parenteral use containing the above canine, feline, or porcine expression vectors. The carrier should be suspended or dissolved in an aqueous solvent, such as a saline solution, and should be present at a concentration of about 0.01-10 mg/ml. A preferred concentration is about 0.1 mg/ml.

FIG. 1: the complete amino acid sequence of dog EPO is given in FIG. 1. The underlined amino acids are the dog signal sequence, while the remaining amino acids are the structural sequence of mature dog EPO.

FIG. 2: the complete amino acid sequence of feline EPO is given in fig. 2. The underlined amino acids are the feline signal sequence, while the remaining amino acids are the structural sequence of mature feline EPO.

FIG. 3: the complete structural sequence of mature porcine EPO is given in fig. 3.

FIG. 4: FIG. 4 shows the results of a study designed to determine the effect of dog EPO encoding DNA administration to dogs. Dogs were divided into groups, one control, and received plasmid DNA encoding human soluble embryo alkaline phosphatase gene (indicated by black diamonds in the figure), and three additional experimental groups. Group A was given 6 doses of 7.5mg EPO DNA in a volume of 25ml (indicated by black boxes in the figure); group B was given 6 doses of 2.5mg EPO DNA (indicated by black triangles in the figure) in a volume of 25 ml; group C received 6 doses of 2.5mg EPO DNA (indicated by X in the figure) in a volume of 3 ml. Each dog in the study was subjected to a 32 day blood count.

FIG. 5: FIG. 5 shows the results of an experiment in which EPO encoding DNA was administered to dogs in a renal failure model. One kidney was surgically removed from 16 dogs and the second kidney was reduced by about 50%. Dogs were bled at 24 hour intervals to induce anemia. After the last bleeding, a total of 12 dogs received DNA encoding dog EPO. Of these, 6 dogs received DNA without transfection-facilitating agent, and the other 6 dogs received DNA complexed with lipid. 4 control dogs were not treated. The percent change in the blood count of each dog was determined for 50 days and the results for the control (black box), animals treated with DNA without transfection-promoting agent (circles, dashed lines) and animals treated with lipid-complexed DNA (circles, dotted lines) are shown.

FIG. 6: this figure also shows the results of the experiments associated with figure 5. Data for control animals and animals treated with lipid-complexed DNA are presented as the change in blood cell count observed about day 12-38 in the study. The fit of the best straight line in relation to these data is also shown. The black circles indicate the results obtained with the lipid-complexed DNA treated animals and the boxes indicate the results with the control animals.

Definition of

The following description uses a number of terms relating to recombinant DNA technology. In order that the specification and claims may be clearly and consistently understood, the following definitions are provided.

Cloning vector: can autonomously replicate in a host cell and is characterized by having onePlasmid, phage DNA or other DNA sequences containing one or a small number of restriction enzyme recognition sites. The foreign DNA fragment can be spliced into the vector at these sites to achieve replication and cloning of the fragment. The vector may contain a marker suitable for identifying transformed cells. For example, the marker may provide tetracycline resistance or ampicillin resistance.

Expression vector: a vector similar to the cloning vector, but capable of inducing expression of the cloned DNA upon transformation of the host. The cloned DNA is often under the control of (i.e., operably linked to) regulatory sequences such as promoters or enhancers. The promoter sequence may be constitutive, inducible or repressible.

Host computer: any prokaryotic or eukaryotic cell that serves as a recipient for an expression vector or cloning vector is a "host" for that vector. Examples of cells which can serve as hosts are well known in the art, and the techniques for cell transformation are likewise well known (see, e.g., Sambrook et al, molecular cloning: A laboratory Manual (R) ("A)Molecular Cloning：A Laboratory Manual) Second edition, cold spring harbor press (1989)). The host cell may be present in vivo and subjected to transfection with nucleic acid for injection into an animal.

Promoters: DNA sequences located in the vicinity of the initiation codon, usually found in the 5' region of the gene. Transcription is initiated from a promoter. If the promoter is of the inducible type, the rate of transcription can be increased with an inducer.

Expression of: expression refers to the process by which a polypeptide is produced from DNA. This process involves transcription from the polypeptide-encoding DNA segment into mRNA and translation of the mRNA into the final polypeptide product.

Preparation of expression vector

The present invention relates to methods of treating anemia in dogs, cats and pigs by administering expression vectors encoding EPO from dogs, cats and pigs, respectively. The full-length amino acid sequences of these types of EPO are shown in FIGS. 1-3. Wen et al (blood, 82: 1507-. MacLeod et al (am. J. ve. Res.) 59: 1144-1148(1998)) also describe methods for isolating and inserting dog EPO into expression vectors. Although these methods can be used to obtain EPO sequences suitable for use in the present invention, many other techniques for isolating the genetic elements described can be used (see, e.g., Sambrook et al, molecular cloning: A laboratory Manual, second edition, Cold spring harbor Press (1989)). Thus, DNA may be chemically synthesized, obtained by screening cDNA libraries, or amplified from cDNA using PCR probes corresponding to known EPO sequence regions. A preferred method is to chemically synthesize the desired EPO nucleotide sequence with a signal sequence-encoding segment at the 5' end. Preferably, a signal sequence homologous to the type of EPO delivered is used (e.g., dog hormone preferably uses a dog signal sequence). However, other signal sequences may be used if desired, for example, a dog signal sequence may be used for a porcine structural sequence. The 5 'end of the region encoding the signal sequence and the 3' end of the EPO structural sequence should also include restriction sequences. These restriction sequences should be selected to match the corresponding sites within the vector that will be used to express EPO. For the preferred VR1012 vector, DNA can be constructed with an EcoRV site at the 5 'end and a BglII site at the 3' end.

The expression vector can be constructed using conventional techniques and should include a promoter operably linked to the DNA element encoding EPO. Transcriptional enhancers and other regulatory elements may also be present. Preferably, transcription is initiated by the CMV (cytomegalovirus) promoter. Examples of other promoters that may be used include the promoter of the mouse metallothionein I gene (Haymer et al, J.Mol.appl.Gen.) 1: 273 (1982); the TK promoter of herpes virus (McKnight, cell, 31: 355-365(1982)), and the SV40 early promoter (Benoist et al, Nature, 290: 304 (1981)).

A number of suitable plasmids for use in the present invention have been described (Botstein et al, Miami Winter Symp 19: 265 (1982); Broach, cell 28: 203 (1982); Bollon et al, J.Clin.Hematol.Oncol.) 10: 39(1980) (ii) a And maniotis, cell biology: general thesis (Cell Biology：A Comprehensive Treatise) Volume three, Academic Press, New York, page 563-608 (1980)). A preferred expression vector, "VR 1012", may be constructed as described by Hartikka et al (human gene therapy (hum. Gene Ther.) 7: 1205-1217 (1996)).

Chemical form of expression vector

Many different drugs have been used to promote DNA uptake by cells in vivo. The present method is suitable for use with all of these drugs, however, the expression vector is preferably administered in the absence of a transfection facilitating agent, or mixed with a cationic lipid. Suitable methods for preparing "naked" DNA are described in U.S.5,703,055. Examples of cationic lipids that can be used are described in U.S. Pat. nos. 5,264,68 and 5,459,127, while examples of cationic liposomes that can be used are found in U.S. Pat. No. 4,897,355. When using transfection facilitating agents, selection should be made with the goal of achieving maximum cellular uptake of the expression vector in the treated animal without causing adverse side effects.

Dosage forms and modes of administration

Any mode of administration suitable for in vivo cell transfection and subsequent expression of functional EPO can be used in the present methods. The expression vector encoding EPO can be administered as the sole active agent or together with other nucleic acids or therapeutically active agents. Although intramuscular injection is preferred, there are other modes that can be used, including intradermal, transdermal, intravenous, intraarterial, intraperitoneal, intradermal, and subcutaneous modes. Vectors can also be delivered to animals by the "gene gun" method.

The expression vector may be incorporated into a medicament manufactured using methods conventional in the art (see, e.g., Remington's Pharmaceutical Sciences, 16 th edition, A. Oslo eds., Easton PA (1980)). Generally, the formulation should be suitable for parenteral administration and will include aqueous solvents such as sterile isotonic saline solution, ethanol, aqueous polyglycol solutions, Ringer's solution, and the like. Generally, the expression vector should comprise about 0.01-10mg/ml of the final composition.

Method of treatment

Animals diagnosed with anemia should typically be administered doses of 0.01-20mg by intramuscular injection in a volume of about 0.5-30 ml. One or more injections may be performed. Thus, animals given several injections can receive a total dose of 100mg or more. One suitable protocol involves three injections of DNA into each hindlimb of the animal, with 30 minutes between each injection. Effective treatment was achieved by 6 injections (25 ml of 2.5mg EPO DNA per administration).

These dosages are only guidelines and the actual dosage selected for an individual animal will be determined by the veterinarian based on the clinical situation. The effectiveness of a given dose can be tested by measuring the blood cell count of treated animals at regular intervals (e.g., once every two weeks). Repeated administrations every three or four months may be necessary, and the frequency may be adjusted to the individual animal's response. Other factors that may affect dosage include the cellular uptake efficiency of the expression vector and the rate at which the promoter and other regulatory elements induce EPO synthesis after transfection.

The method is suitable for any in vivo transfection method already described in the art. Preferred methods are those described in WO 90/11092 and the examples section herein. These and similar methods can be effectively used to treat anemia in dogs and cats, regardless of the cause. Thus, animals will respond to treatment methods whether the anemia is due to chronic renal failure, chemotherapy, or simply aging.

Example 1:preparation of DNA encoding EPO

Based on the sequences given in FIGS. 1 and 2 (including the nucleotides encoding the signal sequence), DNA sequences encoding dog-type and cat-type EPO were synthesized. The synthesized DNA has an EcoRV site at the 5 'end and a BgIII site at the 3' end. The DNA was then cloned into plasmid VR 1012. The plasmid contains several genetic elements, including the CMV promoter, which facilitates expression of the cloned gene in mammalian cells both in vivo and in vitro. Recombinant VR1012 containing the canine EPO gene is hereinafter referred to as "canine EPO DNA", and recombinant VR1012 containing the feline EPO gene is referred to as "feline EPO DNA". Coli cultures and purified by cesium chloride gradient ultracentrifugation or anion exchange chromatography. The quality of the DNA preparation was verified by spectrophotometry and agarose gel electrophoresis.

Example 2:increase in mouse blood count following dog EPO DNA injection

This study involved the use of three groups, each containing 20 mice. The first group was treated with dog EPODNA; the second group uses plasmid DNA without the dog EPO gene; and the third group served as untreated control. Mice were injected three times with DNA per hind limb in the rectus femoris muscle. 25 μ g of DNA dissolved in 50 μ l Phosphate Buffered Saline (PBS) was injected at 30 min intervals. The blood cell count was determined on day 0 before the first treatment and on days 9, 16, 23 and 36 after the treatment. Blood was collected by posterior orbital venipuncture using a heparinized hemacytometer. The blood tube was centrifuged and the blood count was determined using a microcytocount capillary reader. The data collected were as follows:

table 1: statistical pairing analysis of mice injected with dog EPO DNA

	Day 0	Day 9	Day 16	Day 23	Day 36
						Plasmid control versus dog EPO	0.1442	0.0001	0.0001	0.0001	0.1502
Plasmid control vs non-injected control	0.0287	0.0563	0.2141	0.9889	0.7015
						Dog EPO versus non-injected control	0.4502	0.0001	0.0001	0.0001	0.0665

Paired analysis of the data shows that there was a significant increase in the blood count of mice treated with dog EPO DNA compared to mice not injected with the control group until 36 days post-treatment (P value less than 0.05). Mice receiving plasmid DNA without the canine EPO gene showed a significant difference at day 0 compared to non-injected mice. This is considered an experimental abnormality since the animals were not treated at the time of blood collection. At any other time point, there was no significant difference between the two control groups. Two weeks after treatment, a drop in the blood cell count was observed for mice treated with canine EPO DNA, presumably due to the induction of an immune response to canine EPO in the mice.

Example 3:increase in blood cell count of mice injected with feline EPO DNA

Two groups of 5 mice were treated with feline EPO DNA and solvent (PBS, "untreated control"), respectively. Concentrations, volumes and injection methods were as described in example 2. Blood cell counts were determined on day 0 before treatment and on days 7, 14 and 21 after treatment. The results were analyzed statistically and are shown in Table 2.

Table 2: statistical pairing analysis of feline EPO DNA-injected mice

	Day 0	Day 7	Day 14	Day 22
					Feline EPO versus non-injected control	0.9357	0.0456	0.0001	0.0111

Data-paired analysis shown in table 2 shows that there was a significant increase in blood cell counts in mice treated with feline EPO DNA compared to untreated controls at 7, 14, and 21 days post-treatment.

Example 4:increase in dog blood cell count by dog EPO DNA injection

To estimate the amount of dog EPO DNA required to cause an increase in blood cell count in dogs, animals were divided into three experimental groups. Six injections per group were performed following the general procedure described in example 2. The volume and amount of each injection are listed in table 3.

Table 3: conditions of each experimental group in EPO DNA-injected dog study

Experimental group	Number of dogs in the study	Volume of	Amount of DNA	Name (R)
					Group A	4	25ml	7.5mg	Large volume, large dose
Group B	4	25ml	2.5mg	Large volume, small dose
					Group C	4	3ml	2.5mg	Small volume, small dose

Each group had four dogs, two were treated with dog EPO DNA, and two controls were treated with plasmid DNA containing the human Soluble Embryo Alkaline Phosphatase (SEAP) gene. The blood cell count was determined as described above.

Injection of dog EPO DNA resulted in an increase in blood counts lasting over 32 days in all groups, while injection of SEAP DNA had no effect on blood counts. Although statistical analysis was limited by the relatively small number of samples, the percent increase in blood count levels in all groups was considered physiologically relevant. The data show that (i) injection of dog EPO DNA results in an increase in dog blood cell count, which confirms the concept of gene therapy, and (ii) an increase in blood cell count for a longer period of time, which indicates that the treated dogs did not produce an immune response to EPO.

For each animal, both dogs in the "large volume, small dose" group (injected with 15mg 150ml total) showed an increase in blood cell count. In both "large volume, large dose" and "small volume, small dose" groups, one of the two animals responded more than the other. When low responders were treated with "boost" doses of dog EPO DNA under "bulk, low dose" conditions, both dogs exhibited an increase in blood cell counts. This indicates that a more consistent hemacytometer response may be expected by injecting dog EPO DNA under "high volume, low dose" conditions.

Example 5:assessing recovery rates of blood cell counts in nephrectomized and exsanguinated dogs

In an experimental model of renal failure, the effect of dog EPO DNA injection on the rate of recovery from anemia was examined. 16 healthy beagle dogs were anesthetized and their left renal artery, vein and ureter of the kidney were ligated and severed. The left kidney was then mobilized and excised. The renal artery of the right kidney was ligated and cut at the first bifurcation, leaving about half of the remaining right kidney for normal perfusion. Dogs were allowed at least one week of recovery prior to venous bleeding. To cause anemia, nephrectomized dogs were bled three times at 24 hour intervals. At each determined time, an amount of blood equal to two percent of the body weight was removed and a double volume of ringer's lactate solution was immediately infused back. Dog EPO DNA was administered to 12 dogs in the manner described above ("large volume, small dose" conditions). Naked dog EPO DNA was administered to 6 dogs and 6 other dogs were administered dog EPO DNA mixed with lipid (Lipofectamine, Life technologies, Gaithersburg, Md.) at a ratio of 4mg DNA/3ml Lipofectamine. The remaining four dogs were left untreated after nephrectomy and phlebotomy.

All nephrectomized and phlebotomized dogs, the values recorded immediately after phlebotomy, showed rising blood counts. Three of the four dogs in the control group (i.e., dogs not treated with dog EPO DNA) showed rising blood counts that were consistently below baseline values (recorded for dogs prior to nephrectomy and phlebotomy). In contrast, dog blood-ball counts treated with dog EPO DNA rose and stabilized at values comparable to those observed prior to nephrectomy and phlebotomy. This indicates that the increase in blood cell counts in these dogs is due to EPO synthesis of the administered DNA. In summary, the rate of rise of blood counts in dogs treated with Lipofectamine mixed DNA was higher than in dogs treated with naked DNA or untreated dogs. Consistent with these observations, reticulocyte counts were higher in dogs treated with EPODNA from dogs mixed with Lipofectamine than in the control, indicating an increase in production of red blood cells. These results indicate that EPO from the administered DNA causes higher blood cell counts and faster increases in anemia dogs. Although the rate of recovery from anemia was comparable in both dogs treated with naked dog EPO DNA and untreated dogs, the results of the study collectively demonstrate that EPO gene therapy can be successfully applied to dogs.

Example 6:increase in blood cell count in outcrossed mice (CD1) following dog EPO DNA injection

In examples 2 and 3 above, inbred BalB/C mice were injected with 50. mu.l volumes of DNA. In this example, the inbred mouse CD1 was injected with dog EPO DNA in a volume of 25. mu.l. Some injections were performed using sodium phosphate as a solvent (150mM pH7.2-7.4), while others were performed with PBS (Dulbecco's phosphate buffer, Life Technologies Inc. (Life Technologies Inc.), Gaithersburg, Md.).

Table 4: conditions of each experimental group in the study of EPO DNA-injected inbred mice

Group of	DNA dosage per hind limb (μ g)	Volume per injection (μ l)	Solvent(s)	Number of injections per hind limb
					I	100	25	Sodium phosphate	1
II	50	25	Sodium phosphate	1
					III	25	25	Sodium phosphate	1
IV	100	25	PBS	1
					V	50	25	PBS	1
VI	25	25	PBS	1
					VII (Positive control)	100	50	PBS	3
VIII (negative control)	0	25	Sodium phosphate	1

The results obtained demonstrate that DNA-based EPO gene therapy can be successfully applied to an inbred mammalian population. It was also found that DNA dissolved in sodium phosphate caused a higher blood count response relative to DNA dissolved in PBS, and that a 25 μ Ι volume of DNA per hind limb injection was sufficient to cause a significant increase in blood count.

Example 7:increase in blood cell count in "senescent" mice injected with dog EPO DNA

This study relates to an example of an increase in the blood cell count of "senescent" (retired breeder) Balb/C mice.

Table 5: conditions of each experimental group in the study of aging mice injected with EPO DNA

Group of	DNA dosage per hind limb (μ g)	Volume per injection (μ l)	Solvent(s)	Number of injections per hind limb
					I	100	25	Sodium phosphate	1
II (negative control)	0	25	Sodium phosphate	1

The results obtained demonstrate that DNA-based EPO gene therapy can be successfully applied to the treatment of "aging" mammals.

Example 8:increase in feline blood cell count following injection of feline EPO DNA

Cats of 6-8 months of age were injected, spleens were excised the first 25 days, and treated with feline EPO DNA according to the protocol described below. Plasmid DNA was extracted as described above, dissolved in sodium phosphate and injected intramuscularly into the rectus femoris muscle. For negative controls, cats were injected with 150mM, pH7.2-7.4 sodium phosphate. Each group contained 5 cats.

Table 6: conditions of each experimental group in research of cats injected with cat EPO DNA

Group of	DNA dose per animal (. mu.g)	Concentration of DNA (mg/ml)	Total volume per injection	Number of injections per hind limb
					1 (negative control)	0	0	15	1
2	37.5	0.5	15	3
					3	12.5	0.5	12.5	1
4	5	0.5	5	1
					5	1	0.1	5	1
6	20	2	5	1

The results obtained demonstrate that plasmid DNA-based EPO gene therapy can be successfully applied to cats. All EPO DNA treatments caused a significant increase in blood cell counts. In terms of dose, 1mg of DNA (dissolved in 10ml of sodium phosphate at a concentration of 0.1mg/ml, injected in two equal volumes, once per hind limb) was found to cause a significant increase in blood count compared to untreated cats.

Sequence listing

<110>PFIZER PRODUCTS INC.

<120> erythropoietin for treating anemia in domestic and farm animals

<130>PC10485A

<140>

<141>

<160>3

<170>PatentIn Ver.2.0

<210>1

<211>192

<212>PRT

<213> domestic dog

<400>1

Met Gly Val His Glu Cys Pro Ala Leu Leu Leu Leu Leu Ser Leu Leu

1 5 10 15

Leu Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu Ile

20 25 30

Cys Asp Ser Arg Val Leu Glu Arg Tyr Ile Leu Glu Ala Arg Glu Ala

35 40 45

Glu Asn Val Thr Met Gly Cys Ala Gln Gly Cys Ser Phe Ser Glu Asn

50 55 60

Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Thr Trp Lys Arg Met

65 70 75 80

Asp Val Gly Gln Gln Ala Leu Glu Val Trp Gln Gly Leu Ala Leu Leu

85 90 95

Ser Glu Ala Ile Leu Arg Gly Gln Ala Leu Leu Ala Asn Ala Ser Gln

100 105 110

Pro Ser Glu Thr Pro Gln Leu His Val Asp Lys Ala Val Ser Ser Leu

115 120 125

Arg Ser Leu Thr Ser Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala

130 135 140

Met Ser Leu Pro Glu Glu Ala Ser Pro Ala Pro Leu Arg Thr Phe Thr

145 150 155 160

Val Asp Thr Leu Cys Lys Leu Phe Arg Ile Tyr Ser Asn Phe Leu Arg

165 170 175

Gly Lys Leu Thr Leu Tyr Thr Gly Glu Ala Cys Arg Arg Gly Asp Arg

180 185 190

<210>2

<211>192

<212>PRT

<213>Felis catus

<400>2

Met Gly Val Arg Glu Cys Pro Ala Leu Leu Leu Leu Leu Ser Leu Leu

1 5 10 15

Leu Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu Ile

20 25 30

Cys Asp Ser Arg Val Leu Glu Arg Tyr Ile Leu Glu Ala Arg Glu Ala

35 40 45

Glu Asn Val Thr Met Gly Cys Ala Glu Gly Cys Ser Phe Ser Glu Asn

50 55 60

Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Thr Trp Lys Arg Met

65 70 75 80

Asp Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu

85 90 95

Ser Glu Ala Ile Leu Arg Gly Gln Ala Leu Leu Ala Asn Ser Ser Gln

100 105 110

Pro Ser Glu Thr Pro Gln Leu His Val Asp Lys Ala Val Ser Ser Leu

115 120 125

Arg Ser Leu Thr Ser Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala

130 135 140

Thr Ser Leu Pro Glu Ala Thr Ser Ala Ala Pro Leu Arg Thr Phe Thr

145 150 155 160

Val Asp Tnr Leu Cys Lys Leu Phe Arg Ile Tyr Ser Ash Phe Leu Arg

165 170 175

Gly Lys Leu Thr Leu Tyr Thr Gly Glu Ala Cys Arg Arg Gly Asp Arg

180 185 190

<210>3

<211>166

<212>PRT

<213> wild boar

<400>3

Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Ile

1 5 10 15

Leu Glu Ala Arg Glu Ala Glu Asn Val Thr Met Gly Cys Ala Glu Gly

20 25 30

Cys Ser Phe Ser Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe

35 40 45

Tyr Thr Trp Lys Arg Met Asp Val Gly Gln Gln Ala Val Glu Val Trp

50 55 60

Gln Gly Leu Ala Leu Leu Ser Glu Ala Ile Leu Arg Gly Gln Ala Leu

65 70 75 80

Leu Ala Asn Ser Ser Gln Pro Ser Glu Thr Pro Gln Leu His Val Asp

85 90 95

Lys Ala Val Ser Ser Leu Arg Ser Leu Thr Ser Leu Leu Arg Ala Leu

100 105 110

Gly Ala Gln Lys Glu Ala Thr Ser Leu Pro Glu Ala Thr Ser Ala Ala

115 120 125

Pro Leu Arg Thr Phe Thr Val Asp Thr Leu Cys Lys Leu Phe Arg Ile

130 135 140

Tyr Ser Asn Phe Leu Arg Gly Lys Leu Thr Leu Tyr Thr Gly Glu Ala

145 150 155 160

Cys Arg Arg Gly Asp Arg

165

Claims (10)

1. Use of a plasmid DNA expression vector in the preparation of a medicament useful for treating dog anemia, wherein:

a) the plasmid DNA expression vector includes a unique EPO sequence element consisting essentially of nucleotides encoding dog erythropoietin; and is

b) The plasmid DNA expression vector further comprises a promoter element operably linked to the EPO sequence element.

2. The use of claim 1, wherein the expression vector is prepared in the absence of a transfection facilitating agent.

3. The use of claim 1, wherein the expression vector is complexed with a cationic lipid.

4. The use of claim 1, wherein the expression vector is made part of a liposome.

5. The use of claim 1, wherein the expression vector is present in the medicament at a concentration of 0.01-10 mg/ml.

6. The use of claim 1, wherein the expression vector is present in the medicament at a concentration of 0.1 mg/ml.

7. The use of any one of claims 1-6, wherein the medicament is in a dosage form suitable for intramuscular injection.

8. A pharmaceutical composition for parenteral use comprising:

(a) a plasmid DNA expression vector, wherein:

i) the plasmid DNA expression vector includes a unique EPO sequence element consisting essentially of nucleotides encoding dog erythropoietin; and

ii) the plasmid DNA expression vector further comprises a promoter element operably linked to said EPO sequence element; and

(b) an aqueous solvent to dissolve or suspend the expression vector.

9. The pharmaceutical composition of claim 8, wherein the expression vector is present at a concentration of 0.01-10 mg/ml.

10. The pharmaceutical composition of claim 8, wherein the expression vector is present at a concentration of 0.1 mg/ml.