Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
2001-04-23
2003-11-04
Priebe, Scott D. (Department: 1635)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C435S320100, C435S455000, C435S456000
Reexamination Certificate
active
06641807
ABSTRACT:
The present invention relates to the delivery of erythropoietin (EPO) to a mammal. More particularly, the present invention relates to provision of EPO in a mammal by means of expression from encoding nucleic acid included in an expression vector, that is by means of gene therapy. The present invention is based on the inventors' experimental demonstration that therapeutic levels of EPO can be achieved using helper-dependent adenoviral (Hd-Ad) vectors, which levels are far beyond any levels previously attained using a variety of vectors, including adenoviral (Ad) vectors (i.e. non-helper-dependent).
Erythropoietin (EPO) is a protein of great interest because of its therapeutic usefulness in a variety of diseases. As is well known, the gene for human EPO was cloned by Amgen (see e.g. WO85/02610, EP-A-0148605) and recombinantly produced EPO (rEPO) has attained a huge market (in excess of 2.9 billion dollars). Currently, rEPO is administered to patients in protein form.
Despite its success, there is a number of problems with delivery of rEPO resulting in various unmet clinical needs, primarily because of the prohibitive cost of providing sufficient rEPO to achieve a long-term therapeutically effective dosage. Sufferers include individuals with anaemia of Chronic Renal Failure (CRF), anaemias due to beta-thalassaemia, and sickle cell anaemia (SCA). Large numbers of such individuals go untreated despite the fact that good therapeutic results can be achieved as long as enough EPO is provided.
In CRF there is an irreversible decline of kidney function, and the patients manifest a sequel of renal dysfunctions, including anaemia, but do not necessarily require dialysis except at the end stage renal disease (ESRD). At this stage the patient require either regular dialysis or kidney transplant.
CRF patients may be treated with rEPO, such treatment involving starting doses of 50-100 U/Kg, three times weekly, to achieve an increment of at least 5-6 point in the haematocrit. (Note that in vivo bioactivity of EPO is generally determined by the simple measurement of increase in haematocrit (Hct) by centrifugation of heparinsed blood in capillary tubes.) If this is not achieved within 8 weeks, the dose needs to be increased. Maintenance doses need to be individualised (Hct increase over 48%, are possibly deadly), with average of 75 U/Kg three times weekly, but ranging from 12.5 to 525 U/Kg three times weekly.
Circulating rEPO half life is of 4-13 hours if administered i.v., or 25-30 hours after s.c. administration. Over 95% of CRF patients respond well to the treatment, with a measurable Hct increase, and all are reported to become transfusion-independent after 2 months of treatment.
In both beta-thalassaemia and sickle cell anaemia the formation of a normal &agr;
2
&bgr;
2
haemoglobin (Hb) is impaired. Studies in baboons demonstrate that large doses (800-9,000 U/kg i.v.) of recombinant human EPO given i.v. increase gamma-globin chain synthesis and foetal Hb (AL-Khatt et al., N. En. J. Med., 1987, 317: 415-420).
Clinical trials with rEPO in SCA and beta-thalassaemia, with average doses of 500-1500 U/kg, show a rise in RBC count, Hb content and Hct. In SCA a significant increase of foetal Hb correlates with improved quality of RBC, reduced sickling episodes and improved quality of life.
There is no report of the insurgence of anti-EPO antibodies in CRF patients, even those treated for over 4 years, nor in patients with SCA or beta-thalassaemia.
What denies many individuals rEPO treatment is the costs of providing so much: ≧1,500 U/kg is required three times weekly for significant Hct increase and induction of &ggr;-globulin synthesis in SCA patients.
The provision of an effective system for delivery of sufficient EPO would have ready application given the proven therapeutic effectiveness of the protein in diseases such as those discussed above.
The dosage of biologically active circulating EPO required to reach the therapeutic window for Hb-F stimulation is believed to be in excess of 0.900 U/ml (given that >1,5000 U/Kg three times weekly is necessary, and that the half life of EPO administered s.c. is 18 hrs). To date, despite many attempts using numerous approaches, there has been no report of such a level being achieved using a gene therapy approach, i.e. delivery of EPO by means of expression in the body from encoding nucleic acid conveyed within a recombinant vector.
Delivery of EPO cDNA by different means are described in the prior art. The highest level of circulating EPO reported is around 0.75 U/ml (Kessler P. D. et al. 1996, PNAS 93, 14082-7), well below the level required to have beneficial effect in SCA or beta-thalassaemia.
Studies have been published by other laboratories using Adeno vectors carrying the EPO gene which have shown limitations of these vectors in providing appropriate gene dosage and controlled hormonal release over a prolonged period of time.
Descamp et al., (1994, Human Gene Therapy 5:979-985) used an adenovector containing monkey EPO cDNA under the control of an RSV-LTR promoter. A minimum of 5×10
9
viral PFUs (plaque forming units) were required to give Hct increase (observed in a subset of animals). Example 15 below includes a comparison of results achieved using an embodiment of the present invention with results of Descamp et al.
Svensson et al. (1997, Human Gene Therapy 8:1797-1806) used an adenovector containing a mouse EPO coding sequence operably linked to an EF1&agr; promoter, injecting particles i.m. Maximal EPO levels with 10
9
PFUs/mouse was 90 mU/m. No real dose response was shown. The same group earlier published Tripathy et al., 1996 Nat Med., 2: 545-50, and Tripathy et al., 1996 PNAS USA 93, 10876-80, in the latter of which naked DNA was injected into muscle, the maximal EPO levels achieved in blood being 50 mU/ml. The minimal amound of naked DNA needed to observed Hct increase was 10 mg/mouse, 500 mg/kg, corresponding to 35 mg/injection for a human of average 70 kg weight, a huge amount. Example 14 below includes comparison of results achieved using an embodiment of the present invention compared with the results of Svensson et al.
Remarkably, the experimental work described below demonstrates that use of embodiments of the present invention allows for circulating EPO levels of 300 U/ml to be achieved, i.e. far in excess of the level required for effective therapy. Furthermore, these levels can be obtained following a single injection and sustained over long periods of time.
The present invention in various aspects and embodiments employs an EPO encoding sequence within an adenovirus vector in which the entire Adenoviral genome coding sequences have been removed and substituted with exogenous DNA stuffer sequences, generally a Helper-Dependent Adenovirus vector (Hd-Ad).
A general aspect of the present invention provides for the use of such an adenoviral vector in delivery of erythropoietin to an individual. Such delivery is especially at a level in the serum of the individual of at least about 0.005, or 0.1, or 0.5, or 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 U/ml, and may be greater than about 10 U/ml, such as about 20, 30, 40 or 50 U/ml. As noted, levels of up to 300 U/ml are achievable using embodiments of the present invention.
One unit (U) of EPO corresponds approximately to 10 ng of pure protein and can be defined with reference to the international standard WHO-EPO 2nd International Reference Preparation (Annable et al., 1972, Bull. Wld, Hlth. Org. 47: 99) as the amount that is required to produce equivalent [
3
H]-thymidine incorporation into spleen cells from phenylhydrazine-treated mice to that expressed by 1 unit of the WHO standard preparation, or the amount needed to induce 50% of maximum growth (FC50) in erythroleukaemia cells, TF1.
One advantage that may be attained using the present invention is a dose response curve, making it possible to calculate the amount of vector to administer to achieve a desired level of circulating EPO.
One aspect of the present invention provides a method of
Ciliberto Gennaro
La Monica Nicola
Savino Rocco
Giesser Joanne M.
Merck & Co. , Inc.
Priebe Scott D.
Switzer Joan E.
Whiteman Brian
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