METHOD FOR TREATING ACUTE INTERMITTENT PORPHYRIA (AIP) AND OTHER PORPHYRIC DISEASES

Abstract: A method for treatment or prophylaxis of disease caused by deficiency, in a subject, of an enzyme belonging to the heme biosynthetic pathway, the method comprising administering, to the subject, an effective amount of a catalyst which is said enzyme or an enzymatically equivalent part or analogue thereof. The disease is selected from the group consisting of, acute intermittent porphyria (AIP), ALA deficiency porphyria (ADP), Porphyria cutanea tarda (PCT), Hereditary coproporphyria (HCP), Harderoporphyria (HDP), Variegata porphyria (VP), Congenital erythropoietic porphyria (CEP), Erythropoietic protoporphyria (EPP), and Hepatoerythropoietic porphyria (HEP). The catalyst is an enzyme selected from the group consisting of porphobilinogen deaminase (PBGD) ALA dehydratase, Uroporphyrinogendecarboxylase, Coproporphyrinogen oxidase, Coproporphyrinogen oxidise, Protoporphyrinogen oxidase, Uroporphyrinogen III synthase, Ferrochelatase, and Uroporphyrinogen decarboxylase, or an enzymatically equivalent part or analogue thereof. In addition the invention relates to the use of PBGD and to a method of gene therapy.

Description: METHOD FOR TREAΗNG ACUTE INTERMITTENT PORPHYRIA (AIP) AND OTHER PORPHYRIC DISEASES 5 FIELD OF THE INVENTION The present invention relates to novel methods of treating and preventing disease caused by absence or deficiency of the activity of enzymes belonging to the heme biosynthetic pathway. More specifically, the invention pertains to methods of 10 alleviating the symptoms of certain porphyrias, notably acute intermittent porphyria including gene therapy. BACKGROUND OF THE INVENTION 15 Heme biosynthetic pathway Heme is a vital molecule for life in all living higher animal species. Heme is involved in such important processes as oxygen transportation (haemoglobin), drug detoxification (cytochrome P450), and electron transfer for the generation of chemical energy (ATP) 20 during oxidative phosphorylation in mitochondria. Heme is synthesised in eight consecutive enzymatic steps starting with glycin and succinyl-CoA. Sassa S. 1 996, Blood Review, 10, 53-58 shows a schematic drawing of the heme biosynthetic pathway indicating that that the first enzymatic step (ALA- 25 synthetase) and the last three steps (coproporphyrinogen oxidase, protoporphyrinogen oxidase and ferrochelatase) are located in the mitochondrion whereas, the remaining are cytosolic enzymes. Important regulation of the heme biosynthetic pathway is delivered by the end product 30 of the metabolic pathway, namely heme, which exerts a negative inhibition on the first rate-limiting enzymatic step (conducted by ALA-synthetase) in the heme biosynthetic pathway. (Strand et al. Proc. Natl. Acad. Sci. 1970, 67, 1 31 5-1 320). Deficiencies in the heme biosynthetic enzymes have been reported leading to a group of diseases collectively called porphyrias. A defect in the third enzymatic step leads to acute intermittent porphyria, AIP. Acute Intermittent Porphyria Acute intermittent porphyria (AIP) is an autosomal dominant disorder in man caused by a defect (50% reduction of activity) of the third enzyme in the heme biosynthetic pathway, porphobilinogen deaminase, (also known as porphobilinogen ammonia- lyase(polymerizing)), E.C. 4.3.1 .8.(Waldenstrόm J. Acta.Med. Scand. 1937 Suppl.82). In the following, this enzyme will be termed "PBGD". Clinical manifestation of AIP The reduction in enzymatic PBGD activity makes this enzyme the rate limiting step in the heme biosynthetic pathway, with a concomitant increase in urinary and serum levels of delta-aminolevulinic acid (ALA) and porphobilonogen (PBG). The clinical manifestation of AIP involves abdominal pain and a variety of neuropsychiatric and circulatory dysfunctions. As a result of the enzymatic block, heme precursors such as porphobilinogen (PBG) and delta-aminolevulinic acid (ALA) are excreted in excess amounts in the urine and stool. In acute attacks, high levels of PBG and ALA are also found in serum. These precursors are normally undetectable in serum in healthy individuals. The neuropsychiatric disturbances observed in these patients are thought to be due to interference of the precursors with the nervous system or due to the lack of heme. For instance, ALA bears a close resemblance to the inhibitory neurotransmitter 4- aminobutyric acid (GABA) and has been suggested to be a neurotoxin. (Jeans J. et al. American J. of Medical Genetics 1996,65, 269-273). Abdominal pain is the most frequent symptom in AIP patients and occurs in more than 90% during acute attacks, which will be followed rapidly by the development of peripheral neuropathy with weakness in proximal muscles, loss of pinprick sensation, and paraesthesia. Tachycardia, obstipation or diarrhoea may also be present. During acute attacks behavioural changes, confusion, seizures, respiratory paralysis, coma and hallucinations may be present. Hypertension is also associated with AIP, with as high as 40% of patients showing sustained hypertension between attacks. An association between chronic renal failure (Yeung L. et al. 1983, Q J. Med 52, 92-98) and AIP as well as hepatocellular carcinoma. (Lithner F. et al. 1984, Acta.Med. Scand. 21 5,271 -274), has been reported. The AIP is a lifelong disease, which usually becomes manifest in puberty. Factors precipitating acute attacks. Most precipitating factors exhibit an association with the first rate-limiting enzyme in the heme biosynthetic pathway through heme, the final product of the pathway. A lowering of the heme concentration will immediately increase the rate of ALA- synthetase. An overproduction of ALA then makes the partially deficient PBGD enzyme (50% activity) now rate-limiting with an accumulation of the heme precursors ALA and PBG. Drugs that induces cytochrome P450 such as barbiturates, estrogens, sulphonamides, progesterone, carbamyazepine, and phenytoin can all precipitate acute attacks. (Wetterberg L. 1976, In Doss M. Nowrocki P. eds. Porphyrias in Human Disease. Reports of the discussion. Matgurg an der Lahn, 1 91 -202). The clinical manifestation is more common in women, especially at time of menstruation. Endocrine factors such as synthetic estrogens and progesterone are known precipitating factors. A significant factor is also the lack of sufficient caloric intake. Hence, caloric supplementation during acute attacks reduces clinical symptoms. (Welland F.H. et al. 1964, Metabolism, 13, 232). Finally, various forms of stress including illness, infections, surgery and alcoholic excess have been shown to lead to precipitation of acute attacks. There are also cases of acute attacks where no precipitating factor can be identified. Prevalence of AIP Prevalence of 0.21 % has been reported (Tishler P.V: et al. 1985, Am.J. Psychiatry 142,1430-1436), with as high a prevalence as 1 per 1 500 in geographic isolates in northern Sweden (Wetterberg L. 1967, Svenska bokfόrlaget Nordstedt, Stockholm). Prevalence up to 200 per 10,000 inhabitants have been reported from Arjepong in Northern Sweden. (Andersson, Christer, Thesis, 1997, ISBN 91 /7191 /280/0, pp. 22- 23). Existing treatment of AIP The treatment of AIP as well as of other types of porphyrias such as variegata, hereditary coproporphyria, harderoporphyria, and aminolevulinic acid dehydratase deficiency, are basically the same. Existing therapies for AIP, are all aimed at reducing circulating PBG and ALA by inhibiting the first rate-limiting enzymatic step ALA- synthetase. This inhibition of ALA-synthetase is achieved by increasing circulating heme, since heme is a negative feed back regulator of ALA-synthetase. Hematin treatment, high caloric intake or inhibition of heme breakdown by Sn-mesoporphyrin administration are the existing therapies today. These therapies have shown limited efficacy. Treatment between acute attacks involves sufficient caloric intake and avoidance of drugs and immediate treatment of infections. Patients that experience acute attacks are treated with intravenous carbohydrates usually dextrose (300 g/day) and intravenous hematin (3 - 8 mg/(kg day)). Treatments with long acting agonistic analogues of LHRH, have been shown to reduce the incidence of pre-menstrual attacks by inhibiting ovulation in AIP patients. Finally, treatments involving heme analogues Sn-mesoporphyrin, which inhibit heme breakdown have also been attempted. Medical need in AIP The lack of effective treatment for AIP is well recognised. In a US mortality study in AIP patients requiring hospitalisation it was concluded that the mortality rate was 3.2- fold higher as compared to a matched general population. Suicide was also a major cause of death, occurring at a rate of 370 times that expected in the general population (Jeans J. et al.1996, Am. J. of Medical Genetics 65, 269-273). Hematin therapy is usually initiated when high caloric intake is not sufficient to alleviate acute attacks. Studies with hematin have been performed but these studies generally used the patients as their own control after the patients did not respond to high carbohydrate treatment. (Mustajoki et al. 1 989, Sem. Hematol. 26, 1 -9). The one controlled study with hematin treatment reported, failed to reach statistical significance due to too small a patient number (Herrick A.L. et al 1 989, Lancet 1 , 1295-1297). In conclusion, there is a definite need for the provision of novel therapeutic/ prophylactic methods aimed at these diseases. DISCLOSURE OF THE INVENTION Levels of ALA and PBG found in urine in patients with symptomatic AIP, are in the range of 1 -203 mg/day and 4-782 mg/day, respectively. Normal excretion of ALA and PBG is very low (0-4 mg/day). Important is the observation that these patients also have elevated levels of ALA and PBG in serum. It was shown in a study that AIP patients had significantly elevated levels of ALA (96 μg %) and PBG (334 μg %) in serum in connection with acute attacks and that the severity of the attacks were correlated to high levels of ALA and PBG. Hence, it is important to reduce the circulating levels of ALA and PBG in order to eliminate clinical symptoms and to normalize the heme pool. The present inventors present a new therapeutic rational in the treatment of AIP, a rationale using PBGD, preferably recombinant PBGD (rPBGD) in order to reduce circulating high levels of PBG in serum by metabolising (by enzymatic conversion) PBG to hydroxymethylbilane (HMB), which is the normal product of the reaction. This substitution therapy will lead to a normalization of PBG in serum as well as to a normalization of the heme pool. It will also lead to a normalization of ALA in serum, since these heme precursors are in equilibrium with each other. A lowering of serum ALA and PBG is expected to result in a concomitant relief of symptoms. The product of the reaction (HMB) will diffuse back into the cells and enter the normal heme biosynthetic pathway and will become subsequently metabolized to heme. Hence, PBGD administered by injections will carry out its normal catalytic function by converting PBG to HMB in serum (extracellularly, not inside the cells), where it normally functions. The new therapeutic idea is based on the assumption that ALA, PBG and HMB permeate cellular membranes or are transported specifically across them. An alternative to this is to administer a form of PBGD which will be able to act intracellularly, either as a consequence of formulation or as consequence of modification of PBGD so as to facilitate its entry into cells from the extracellular compartment. The observation that AIP patients have large amounts of these heme precursors in the serum supports the idea that PBG does not accumulate intracellularly, but is released from the cells into serum when the intracellular concentration increases due to the PBGD enzymatic block. The basic new therapeutic concept for AIP is valid for all porphyrias and therefore the invention is in general aimed at treating these diseases by substituting the reduced or missing enzymatic activity characterizing the porphyrias. Hence, in its broadest aspect, the invention pertains to a method for treatment or prophylaxis of disease caused by deficiency, in a subject, of an enzyme belonging to the heme biosynthetic pathway, the method comprising administering, to the subject, an effective amount of a catalyst which is said enzyme or an enzymatically equivalent part or analogue thereof. Alternatively investigations in treating the porphyrias have also suggested gene therapy, thus aiming at introducing genetic material in relevant cells, which will then take over the in vivo production of the enzyme of interest. Hence, by the term "catalyst" is herein meant either the relevant enzyme which is substituted as it is, or an enzymatically equivalent part or analogue thereof. One example of an enzymatically equivalent part of the enzyme could be a domain or subsequence of the enzyme which includes the necessary catalytic site to enable the domain or subsequence to exert substantially the same enzymatic activity as the full- length enzyme or alternatively a gene coding for the catalyst. An example of an enzymatically equivalent analogue of the enzyme could be a fusion protein which includes the catalytic site of the enzyme in a functional form, but it can also be a homologous variant of the enzyme derived from another species. Also, completely synthetic molecules which mimic the specific enzymatic activity of the relevant enzyme would also constitute "enzymatic equivalent analogues". In essence, the inventive concept is based on the novel idea of substituting the reduced enzymatic activity in the subject simply by administering a catalyst which will "assist" the enzyme which is in deficit. The precise nature, however, of the catalyst is not all-important. What is important is merely that the catalyst can mimic the enzymatic in vivo activity of the enzyme. The term "the heme biosynthetic pathway" refers to the well-known enzymatic steps (cf. e.g. Sassa S. 1 996, Blood Review, 10, 53-58) which leads from glycin and succinyl-CoA to heme, and enzymes belonging to this synthetic pathway are porphobilinogen deaminase (PBGD), ALA dehydratase, Uroporphyrinogendecarboxylase, Coproporphyrinogen oxidase, Coproporphyrinogen oxidise, Protoporphyrinogen oxidase, Uroporphyrinogen III synthase, Ferrochelatase, and Uroporphyrinogen decarboxylase. Hence, in line with the above, a catalyst used according to the invention is such an enzyme or an enzymatically equivalent part or analogue thereof. It should be noted that all of the above-mentioned enzymes have been sequenced, thus allowing recombinant or synthetic production thereof. The diseases related to reduced activity of these enzymes are acute intermittent porphyria (AIP), ALA deficiency porphyria (ADP), Porphyria cutanea tarda (PCT), Hereditary coproporphyria (HCP), Harderoporphyria (HDP), Variegata porphyria (VP), Congenital erythropoetic porphyria (CEP), Erythropoietic protoporphyria (EPP), and Hepatoerythropoietic porphyria (HEP). By the term "effective amount" is herein meant a dosage of the catalyst which will supplement the lack or deficiency of enzymatic activity in a subject suffering from porphyria caused by reduced activity of one of the above-mentioned enzymes. The precise dosage constituting an effective amount will depend on a number of factors such as serum half-life of the catalyst, specific activity of the catalyst etc. but the skilled person will be able to determine the correct dosage in a given case by means of standard methods (for instance starting out with experiments in a suitable animal model such as with transgenic animals so as to determine the correlation between blood concentration and enzymatic activity). The disease which is the preferred target for the inventive method is AIP, and therefore the catalyst is PBGD or an enzymatically equivalent part or analogue thereof. It is most preferred that the catalyst is a recombinant form of the enzyme belonging to the heme biosynthetic pathway or of the enzymatically equivalent part or analogue thereof, since recombinant production will allow large-scale production which, with the present means available, does not seem feasible if the enzyme would have to be purified from a native source. Preferred formulations and dosage forms of the catalyst are exemplified for, but not limited to, PBGD in the detailed description hereinafter, and these formulations also are apparent from the claims. It will be appreciated that these formulations and dosage forms are applicable for all catalysts used according to the invention. One important embodiment of the method of the inventions is one wherein the catalyst, upon administration, exerts at least part of its enzymatic activity in the intracellular compartment. This can e.g. be achieved when the catalyst is an enzymatically equivalent part or analogue of the enzyme, since such variations of the enzyme can be tailored to render them permeate cell membranes. Hence, when the catalyst is a small artificial enzyme or an organic catalyst which can polymerize porphobilinogen to hydroxymethylbilane, it should be possible for the skilled man to introduce relevant side chains which facilitates entry into the intracellular compart- ment. Alternatively, the catalyst is the enzyme, but formulated in such a manner that it exerts at least part of its enzymatic activity intracellularly upon administration to the subject. This can be achieved by tagging the enzyme with specific carbohydrates or other liver cell specific structures for specific liver uptake, i.e. the enzyme (or analogue) is modified so as to facilitate active transport into e.g. liver cells. Although the above embodiments are interesting, it is believed that the normal, practical embodiment of the invention will involve use of a catalyst which exerts substantially all its enzymatic activity extracellularly in the bloodstream, since it is believed that the metabolic products of the enzymatic conversion of the relevant heme precursor will permeate freely into the intracellular compartment where the remaining conversions of the heme biosynthetic pathway can take place. Alternatively, the metabolic product may be excreted from the subject via urine and/or faeces at least to some extent. As mentioned above, it is preferred that the catalyst is produced recombinantly, i.e. by a method comprising a) introducing, into a suitable vector, a nucleic acid fragment which includes a nucleic acid sequence encoding the catalyst; b) transforming a compatible host cell with the vector; c) culturing the transformed host cell under conditions facilitating expression of the nucleic acid sequence; and d) recovering the expression product from the culture and optionally subjecting the expression product to post-translational processing, such as in vitro protein refolding, enzymatic removal of fusion partners, alkylation of amino acid residues, and deglycosylation, so as to obtain the catalyst. For relatively small catalysts (e.g. those constituted mainly of the active site of the enzyme), the catalyst can alternatively be prepared by liquid-phase or solid-phase peptide synthesis. A more detailed explanation of the recombinant production of the model enzyme PBGD is given in the detailed section hereinafter, but as mentioned herein the same considerations apply for all other peptide catalysts of the invention. One of the main advantages of producing the catalyst by recombinant or synthetic means is, that if produced in a non-human cell, the catalyst is free from any other biological material of human origin, thus reducing problems with known or unknown pathogens such as viruses etc. The dosage regimen will normally be comprised of at least one daily dose of the catalyst, (preferably by the intravenous route). Normally 2, 3, 4 or 5 daily dosages will be necessary, but if sustained release compositions are employed, less than 1 daily dosage are anticipated. The daily dosage should be determined on a case by case basis by the skilled practitioner, but as a general rule, the daily dosage will be in the range between 0.01 - 1 .0 mg/kg body weight per day of the catalyst. More often the dosage will be in the range of 0.05 - 0.5 mg/kg body weight per day, but it should never be forgotten that precise dosage depends on the dosage form and on the activity of the catalyst as well as on the degree of deficiency of the relevant enzyme and an individualized treatment, where the dose is adjusted to normalize patient serum and urine precusor levels. The most correct way of determining the correct dosage is based on the patient specific precursor levels. The precursor being the product of the enzymatic reaction. For PBGD, the daily dosage is about 0.08-0.2 mg per kg body weight per day, and most often 0.1 mg per kg body weight per day will be the dosage of choice. It is believed that comparable dosages will be applicable for the other full-length enzymes. Finally, as will be appreciated from the above disclosure, the invention is based on the novel idea of providing substitution for the enzymes lacking in activity. To the best of the knowledge of the inventors, therapeutic use of catalysts having such effects have never been suggested before, and therefore the invention also pertains to a catalyst as defined herein for use as a pharmaceutical. Furthermore, use of such catalysts for the preparation of pharmaceutical compositions for treatment of the above-discussed diseases is also part of the invention. Legends to figures: Figure 1 : Cicular map of PBGD clone # 1 .1 in pBluescript SK Figure 2: Circular map of expressed plasmid pExpO Figure 3: Circular map of expressed plasmid pPBGD 1 .1 Figure 4: Circular map of expressed plasmid pKK223-3 Figure 5: Circular map of expressed plasmid pExpl Figure 6: Circular map of expressed plasmid pPBGD 1 .1 Tra Figure 7: Circular map of expressed plasmid pExp1 -M2 Figure 8: Circular map of expressed plasmid pExp1 -M2-Puc-BB Figure 9a-9x: PBGD clone # 1 .1 in pBluescript SK - Sequence DETAILED DISCLOSURE OF THE INVENTION In a first embodiment the invention relates to a method for treatment or prophylaxis of disease caused by deficiency, in a subject, of an enzyme belonging to the heme biosynthetic pathway, the method comprising administering, to the subject, an effective amount of a catalyst which is said enzyme or an enzymatically equivalent part or analogue thereof. The disease may be selected from the phorphyria group and the catalyst may be an enzyme selected from the group consisting of porphobilinogen deaminase (PBGD) ALA dehydratase, Uroporphyrinogendecarboxylase, Coproporphyrinogen oxidase, Coproporphyrinogen oxidise, Protoporphyrinogen oxidase, Uroporphyrinogen III synthase, Ferrochelatase, and Uroporphyrinogen decarboxylase, or an enzymatically equivalent part or analogue thereof. In a preferred embodiment, the disease is AIP and the enzyme is PBGD or an enzymatically equivalent part or analogue thereof. In a further embodiment, the catalyst is a recombinant form of the enzyme belonging to the heme biosynthetic pathway or of the enzymatically equivalent part or analogue thereof. The catalyst may be administered by a route selected from the group consisting of the intravenous route, the intraarterial route, the intracutaneous route, the subcutaneous route, the oral route, the buccal route, the intramuscular route, the anal route, the transdermic route, the intradermal route, and the intratechal route. The catalyst is preferrable formulated in an isotonic solution, such as 0.9% NaCI and 10-50 mM Sodium phosphate pH 7.0 +/- 0.5 up to pH 8.0 or Sodium phosphate, glycine, mannitol or the corresponding potassium salts. The catalyst may also be lyophilised, sterile filtered, and in a further embodyment formulated as lipid vesicles comprising phosphatidylcholine or phosphatidylethanolamine or combinations thereof. In a still other embodiment the catalyst is incorporated into erythrocyte ghosts. Also a sustained release formulation may be perforrmed involving biodegradable microspheres, such as microspheres comprising polylactic acid, polyglycolic acid or mixtures of these. A further method according to the invention is wherein the catalyst is lyophilized in a two-compartment cartridge, where the catalyst will be in the front compartment and water for reconstitution in the rear compartment. The two compartment cartridge may be combined with an injection device to administer the catalyst either by a needle or by a needle-less (high pressure) device. It may also be very convenient to administer the catalyst in a formulation of a physiological buffer containing an enhancer for nasal administration. Other formulations for the catalyst include an oral formulation containing lipid vesicles, such as those comprising phospatidylcholine, phosphatidylethanolamine, or sphingomyeline, or dextrane microspheres. The formulation is preferable one which is able to enhance the half-life of the catalyst in the subject's bloodstream. This may by use of a formulation wherein the catalyst has a polyethylene glycol coating. The catalyst may also be complexed with a heavy metal. In a further aspect the catalyst is an enzymatically equivalent part or analogue of the enzyme and exerts at least part of its enzymatic activity intracellularly upon administration to the subject. This may be when the catalyst is a small artificial enzyme or an organic catalyst which can polymerize porphobilinogen to hydroxymethylbilane. Furthermore, the catalyst may be said enzyme formulated in such a manner that it exerts at least part of its enzymatic activity intracellularly upon administration to the subject. In addition the catalyst may be tagged with specific carbohydrates or other liver cell specific structures for specific liver uptake. In a furhter aspect the catalyst exerts substantially all its enzymatic activity extracellularly in the bloodstream. In a still further aspect, the enzymatic activity of the catalyst on its relevant heme precursor results in a metabolic product which 1 ) either moves into the intracellular compartment and is converted further via the remaining steps of the heme biosynthetic pathway or 2) is excreted from the subject via urine and/or faeces. A furhter embodiment of the invention relates to a method wherein the catalyst has been prepared by a method comprising a) introducing, into a suitable vector, a nucleic acid fragment which includes a nucleic acid sequence encoding the catalyst; b) transforming a compatible host cell with the vector; c) culturing the transformed host cell under conditions facilitating expression of the nucleic acid sequence; and d) recovering the expression product from the culture and optionally subjecting the expression product to post-translational processing, such as in vitro protein refolding, enzymatic removal of fusion partners, alkylation of amino acid residues, and deglycosylation, so as to obtain the catalyst. The catalyst may be prepared by liquid-phase or solid-phase peptide synthesis and it is preferable free from any other biological material of human origin. As mentioned above the catalyst may be administered at least once a day, such as 2, 3, 4, and 5 times daily depending on the specific treatment regimen outlined for the patient in that precursor levels for each patient are measured before and/or during treament for evaluation of the specific dosage. Accordingly the daily dosage may be in the range of 0.01 - 1 .0 mg/kg body weight per day, such as in the range of 0.05 - 0.5 mg/kg body weight per day. And the present invention also relates to the use of the catalyst for the preparation of a pharmaceutical composition. It is estimated that a dosage will often be about 0.1 mg per kg body weight per day. Accordingly, the invention also relates to a catalyst which is an enzyme of the heme biosynthetic pathway or an enzymatically equivalent part or analogue thereof, for use as a medicament. Thus in a further embodyment, the invention relates to a catalyst which is an enzyme of the heme biosynthetic pathway or an enzymatically equivalent part or analogue thereof for the preparation of a pharmaceutical composition for the treatment or prophylaxis of diseases caused by deficiency of said enzyme. Naturally, the catalyst may be a recombinant form of the enzyme. An example is a recombinant human PBCD based on any of Seq. ID NO 1 (clone PBGD 1 .1 ) and Seq. ID NO 12 (non-erythro PBGD 1 .1 .1 ). In a preferred embodiment and as will be disclosed in detail below, the invention also relates to a method for treating a patient having af mutation in the PBGD gene causing an enzyme defect, the method comprising use of a human PBGD cDNA sequence of either non-erythropoitic form or erythropoitic form according to the tissue in which PBGD should be expressed, and transfecting the patient with the relevant cDNA Preferably the enzyme defiency is selected from enzyme defiencies resulting in a disease selected from Acute Intermittent Porphyria, (AIP), ALA deficiency porphyria (ADP), Porphyria cutanea tarda (PCT), Hereditary coproporphyria (HCP), Harderoporphyria (HDP), Variegata porphyria (VP), Congenital erythropoietic porphyria (CEP), Erythropoietic protoporphyria (EPP), and Hepatoerythropoietic porphyria (HEP). In a preferred embodiment, the human PBGD cDNA sequence is selected from Seq. ID NO 1 (clone PBGD 1 .1 ) and Seq. ID NO 12 (non-erythro PBGD 1 .1 .1 ). The transfection may be by use of a vector selected from adenovirus, retrovirus and associated adenovirus. The PBGD gene transfer vector into human cells (erythropoeitic and/or non-erythropoietic) preferable results in normal PBGD activity or in an activity wherin the patient is free of symptoms of disease. A further method of gene therapy treatment of patients with Acute Intermittent Porphyria (AIP) according to the invention is by a correction of one of the specific point mutations identified causing AIP by use of chimeraplasty gene repair. This involve specific designed oligonucletides and a specific knowledge of both the mutation to be corrected and to the sequence on both sides on the mutation. In a specific embodiment of chimeraplasty gene repair is by use of a delivery system for transfection by use of non-viral vectors formulated in a vehicle preparation comprisng one or more components selected from cationic phospholipids, phospholipids, phospholipids mixed with neutral lipids, lictosylated PEI, liposomes liposomes comprising mixtures of natural phopholipids and neutral lipids. The mutation may be selected from the mutations shown in Table A. The following description of preferred embodiments of the invention will focus on recombinant production of PBGD and formulations and uses thereof. It will be appreciated, however, that all disclosures relating to this polypeptide apply also for the other enzymes mentioned above. Hence, production and use of PBGD only exemplifies the invention, but all other enzymes of the heme biosynthetic pathway can substitute PBGD in the embodiments described hereinafter. Production of recombinant PBGD As mentioned above, it is preferred to administer recombinant versions of the various enzymes of the heme biosynthetic pathway. In the following will be described recombinant production of one of these enzymes, namely PBGD. The gene for the erythropoietic PBGD, which is located in the human genome in the chromosomal region 1 1 q 24, is composed of 15 exons spanning 10 kb of DNA and is shown in Grandchamp B. et al. 1 996. J. of Gastroenerology and Hepatology 1 1 , 1046-1052. The gene coding the erythropoietic PBGD enzyme (344 amino acids) (Raich N. et al 1986, Nucleic. Acid. Res, 14, 5955-5968), will be cloned from a human erythropoietic tissue by reverse transcriptase or amplification by PCR (polymerase chain reaction) of the PBGD coding region. The gene will be inserted in a plasmid and transformed into a suitable host cell (a bacterium such as E. coli and B. subtilis, a fungus such as Saccharomyces, or a mammalian cell line, such as CHO cells). The expression of the PBGD gene will be regulated by a promoter which is compatible with the selected host cell. For bacterial production: An endogenous ATG sequence is located at the NH2-terminal end of the rPBGD structural gene for initiation of translation and cytoplasmic expression. Alternatively insert in front of the PBGD coding region a bacterial signal sequence for example an E. coli periplasmic enzyme signal peptide or a signal peptide from a secreted enterotoxin or endotoxin in E. coli, to obtain secretion in E. coli. The plasmid used for production of rPBGD in E. coli was constructed in the following way: Construction of the rPBGD production plasmid Introduction: The erythropoietic expressed form of porphobilinogen deaminase (PBGD) ( Raich N, et al Molecular cloning and complete primary sequence of erythrocyte porphobilinogen deaminase. Nucleic Acids Research 1986 14(15): 5955-67) was cloned and sequence determined. Two forms of PBGD are known. The erythropoietic form is expressed specifically in erythroid progenitors and the constitutive form is expressed in all cells (Grandchamp B, et al Tissue-specific expression of porphobilinogen deaminase. Two isoenzymes from a single gene. Eur J Biochem. 1987 Jan; 162(1): 105-10. mp et. al. 1987.) The two are expressed from the same gene and are identical except for the addition of 17 amino acids at the amino terminus of the constitutive form through alternative exon usage. It was decided to clone and express the erythropoietic form. There are three sequences for PBGD in the Genebank, the two isoforms mentioned above and the genomic sequence (Yoo HW, et al Hydroxymethylbilane synthase: complete genomic sequence and amplifiable polymorphisms in the human gene. Genomics 1993 Jan; 15(1):21-9). These all have nucleotide differences translating to amino acid changes. Before choosing a specific sequence to be expressed for a human therapeutic it was therefore necessary to determine what is the wild type allele. To accomplish this, PBGD cDNA clones were isolated and sequenced from a number of sources to define the most common amino acid usage. Oligonucleotide primers were designed to amplify the coding region from cDNAs by Polymerase Chain Reaction (PCR) (Saiki RK, et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985, Dec 20; 230(4732): 1350-4). These were used to isolate cDNAs from 5 sources of mRNA which were then cloned into a plasmid vector. Eight of these clones were sequenced and along with the published sequences define a wild type allele, which should be the most common amino acid sequence in the population. This wild type allele will be used for protein expression. Strategy: A nested PCR strategy was devised to clone PBGD. The first primer set, (see Table 1 ) lco379 and lco382, are 20mers that bind to sequence outside of the coding region. lco379 is specific for the 5' untranslated region of the mRNA (cDNA) of the erythropoietic form of PBGD. The binding site is in an exon region not expressed in the constitutive form of the enzyme. lco382 binds to the 3' untranslated region of both forms of PBGD. Internal to these are a second set of oligonucleotide primers to be used for the second round of PCR, lco375 and lco376, designed to distal ends of the PBGD coding region. lco375 has 22 bases of sequence homologous to the 5' end of the coding region of the erythropoietic form of PBGD with the ATG start codon followed by an EcoRI endonuclease cleavage site for cloning of the PCR product and 4 bases of sequence to ensure efficient restriction. lco376 has 33 bases homologous to the 3' end of the PBGD coding region with 3 bases changed internally to introduce a Munl/Mfel endonuclease cleavage site through silent mutations and ending with the TAA stop codon. This restriction site will be used to easily introduce sequence encoding a His-Tag to the DNA with oligonucleotide adapters or to enable other 3' modifications. Following the stop codon is a second stop codon to ensure good termination of translation and a Hindlll endonuclease cleavage site for cloning the PCR product followed by 4 bases to ensure efficient restriction. The EcoRI and Hindlll endonuclease cleavage sites introduced onto the ends of the PBGD PCR product ligate into the respective unique restriction sites in the high copy number pBluescriptll SK- (Stratagene) vector for sequencing and will then be used to move the PBGD DNA into an E. coli expression vector for production of recombinant human porphobilinogen deaminase, rh-PBGD. PCR: Six cDNAs were used as a PCR source; spleen, bone marrow, lymph node, lung, whole brain and adipose tissue each from a different pool of human donors (produced by Donald Rao using BRL Superscript II with 500 ng Clontech poly-A RNA in 20 μl reaction volumes per manufacturers instructions except adipose which was made from 5 μg of Clontech total RNA from a single donor). List of equipment and supplies used (see lists below). One μl of each cDNA (approximately 25ng) was amplified with Advantage cDNA polymerase mix (Clontech) with 0.2mM dNTP (PE/ABI) and 0.3μM each of lco379 and lco382 in 50 μ\ reaction volumes. Two cycle PCR was used, with an initial heat denaturation step at 94°C for 1 ' 40" then 28 cycles of 96°C for 16" and 68 °C for 2'. A final extension of 6' at 74°C ensured that extension products were filled out. One fifth of the reaction was run out on a 1 .2% agarose gel with 2 μl of 6X ficol loading dye in 0.5X TBE buffer (Maniatis T., E.F. Fritsch, J. Sambrook. Molecular Cloning (A laboratory Manual) Cold Spring Harbor Laboratory. 1 982 ). The predicted band of 1 .1 kb. was observed by ethidium bromide staining with all sources but lung tissue cDNA. These bands were excised and DNA was isolated with Microcon-30 with micropure inserts (Amicon/Millipore) per manufacturers instructions and buffer exchanged with dH20. One tenth of the recovered DNA was amplified with Advantage cDNA polymerase mix (Clontech) with 0.2mM dNTP (PE/ABI) and 0.3μM each of the internal nested oligonucleotides (lco375 and lco376) at 0.3μM in 50μl reactions. Two cycle PCR was used again with an initial heat denaturation step at 94°C for 1 ' 40" then 2 cycles of 96°C for 16" and 68°C for 2' then 13 cycles of 96°C for 16" and 72°C for 2' with a final extension of 6' at 74°C. Ten μ\ of the 50 μ\ reactions were run on a 1 .2% agarose gel with 2 μ\ 6X loading dye. The resulting bands were of the expected size, 1 .05 kb. The remainder of the PCR reactions were passed through Chromaspin-400 columns (Clontech) per manufacturers instructions to remove reaction components and to exchange buffer with TE (10mM Tris-HCI pH8.0/ 1 mM EDTA). The DNA containing eluates were washed with dH20 and concentrated with Microcon-100 spin-filters (Amicon/Millipore) per manufacturers instructions. Cloning: The purified PBGD DNA was digested for 6 hours with 40 Units each of EcoRI and Hindlll in EcoRI "U" buffer (New England Biolabs (NEB)) in 50 μl reactions at 37°C. Enzymes were heat killed for 20 minutes at 68°C and reactions were spun in Microcon 100 spin-filters to remove small DNA end pieces, washed with dH20 and concentrated. One fifth of the resulting DNA was ligated with approximately 50 ng EcoRI and Hindlll digested and twice gel purified pBluescriptll SK- (Stratagene) and 200 units T4 DNA ligase (NEB cohesive end units) for 15 hours at 16°C. The ligase 5 was heat killed at 75 °C for 10 minutes. The reactions were then buffer exchanged with dH20 and concentrated in Microcon-100 spin filters and volumes taken up to 5 μl with dH20. One μl each was electroporated into 25 μl DH10B Electromax cells (Gibco/BRL) at 2.5Kv/2000hms/25μF in 0.1 cm cuvets with a BioRad electroporator. One ml of SOC medium (Gibco/BRL) was added and the cells were outgrown at 37 °C 10 for one hour at 250 rpm. Cells were plated out on LB plates (Maniatis T., E.F. Fritsch, J. Sambrook. Molecular Cloning (A laboratory Manual) Cold Spring Harbor Laboratory. 1982) with 150 μg/ml ampicillin. The efficiency of all five were approximately twice background (vector ligated without insert.) Colony PCR was used to analyze 18 transformants of each electroporation for the presence of PBGD. An internal PBGD 15 specific primer (lco381 ) was used with a pBluescript specific primer (lco385) to both confirm identity and proper orientation in the vector. The 25 μl reactions were set up on ice to inactivate proteases with primer concentrations of 0.4μM, 0.125U Taq polymerase (Fisher), and 0.2mM dNTP(PE/ABI.) Two cycle PCR was used, with an initial heat denaturation step at 94°C for 1 ' 40" a further denaturing step at 96°C for 20 20 seconds, then 30 cycles of 96°C for 16" and 68°C for 1 ' with a final extension of 4' at 74°C. Five μl of 6X loading dye was added and 1 2.5 μl each were run out on a 1 .2% agarose gel. Results are as follows: 1 2/18 positive colonies for spleen; 10/18 for bone marrow; 8/18 for lymph node; 9/18 for brain and 10/18 for adipose tissue. Two positive colonies each for the first 3 and 1 each for the latter two were grown up in 25 25 ml. liquid LB culture with 150 μg/ml ampicillin over night at 37°C with 250 rpm. Plasmid DNA was purified from the cultures with Qiagen's Tip-100 DNA purification kit per manufacturer's instructions. UV absorbance at 260nm was used to determine the plasmid yields which varied from between 131 and 169 g of highly purified DNA. 30 Sequencing: Sequencing reactions of double stranded plasmid DNA with Big Dye terminator cycle sequencing were performed in a 9700 thermocycler (Perkin Elmer/Applied Biosystems.) Two vector primers (lco383 and lco385) and two PBGD specific internal primers (Ico380 and lco381 ) were used for all 8 plasmids (see sequence). In addition a fifth vector primer (lco285) was used for the brain and adipose derived clones. Reaction conditions were per manufacturers protocol as follows: 500 ng plasmid DNA and 4 pmol oligonucleotide primer with 8 μl ready mix in 20 μl volumes with 30 cycles of 96°C for 12" and 60°C for 4'. Extension products were purified by isopropanol precipitation. To each reaction 20 μl of dH20 and 60 μl Isopropanol were added. These were mixed by inversion and allowed to sit at room temperature for 1 5 minutes then spun for 40' at 3250 rpm in a Beckman GS-6KR centrifuge with the GH3 rotor and Microplate + carriers. Reactions were inverted then spun at 1680 rpm for V to remove liquid from the pelleted DNA. DNA sequence analysis was performed at the University of Washington Biochemistry Department sequencing Laboratory with an Applied Biosystems 377 sequencer. Analysis: The inserts of all 8 clones were confirmed to be PBGD by complete double strand sequence analysis (see sequences 1 - 8). Each has some change(s) from the published sequences. Some changes are unique and some are shared with other clones (see Table 2 and Table 3). For differences found only in one clone, it is difficult to distinguish between PCR or cloning artifacts and actual allelic variations without additional sampling. However, when the same base difference is found in more than one sequence it is unlikely to be from cloning errors. From the alignment of all 1 1 PBGD sequences a set of common bases emerged, the consensus or wild type allele sequence. Five of the eight clones (1 .1 , 1 .3, 2.1 , 3.3, and 5.3.) have the wild type amino acid sequence. Within this set with wild type amino acid sequence, there is only one difference at the nucleic acid level. At position 555, 4 of the 5 sequences have a dGTP while 1 along with the published erythropoietic and genomic PBGD have a dTTP. These appear to be two common alleles, which result in no amino acid difference. There are 2 base changes between clone number 1 .1 and the published erythropoietic PBGD. An adenine to guanine change at base 513 (Leu 171 ) is a silent mutation, which is also present in 9 out of the 1 1 sequences, compared. The second difference is a cytosine to adenine substitution at base 995 (Thr 332.) This is not a silent change, with a threonine to asparagine non-conservative mutation. It appears however that the difference is an error in the published erythropoietic PBGD sequence since all 10 other sequences have an adenine at this position. In addition to these natural variations, there are three additional silent mutations introduced during the cloning at positions 1017, 1018 and 1020 to create a Mun-I site for future manipulations. The PBGD gene was ligated into pBluescript SK plasmid generating the pSK-PBGD 3988 bp plasmid, which was sequenced (see Fig. 1 , Fig. 9a-9x and sequence 9). Conclusion: For any recombinant therapeutic protein it is important that the wild type allele be used to reduce the potential for immunogenicity. We feel confident through our survey of the literature and analysis of PBGD sequence from different individuals that clone number 1 .1 represents the most prevalent "wild type" allele in the population with respect to amino acid sequence. Clone number 1 .1 contains the consensus wild type amino acid sequence and differs from the published erythropoietic PBGD sequence by only one amino acid. Because this difference is found in all the other PBGD clones besides the erythropoietic PBGD sequence, it, rather than the published erythropoietic sequence, is deamed to be the prevalent wild type sequence. For this reason PBGD encoded by clone number 1 .1 was chosen for production of recombinant human porphobilinogen deaminase (rh-PBGD.) Table 1 Oligonucleotide primers: lco375-pbgds (32 mer) coding region 5' end w/ EcoRI site sense 5' CGT GGA ATT CAT GAG AGT GAT TCG CGT GGG TA 3' lco376-pbgda (47 mer) coding region 3'end w/ Hindlll site antisense 5' GGA GAA GCT TAT TAA TGG GCA TCG TTC AAT TGC CGT GCA ACA TCC AG 3' lco378-csnonc (20 mer) constitutive form non-coding sense 5' TCC AAG CGG AGC CAT GTC TG 3' lco379-esnonc (20 mer) erythropoietic form non-coding sense 5' TCG CCT CCC TCT AGT CTC TG 3' Ico380-sinter (21 mer) internal coding sense 5' CAG CAG GAG TTC AGT GCC ATC 3' lco381-ainter (21 mer) internal coding antisense 5' GAT GGC ACT GAA CTC CTG CTG 3' lco382-anonc (20 mer) non-coding sense 5' CAG CAA CCC AGG CAT CTG TG 3' lco383-pSKT7 (22 mer) pBluescript T7 promoter 5' GTA ATA CGA CTC ACT ATA GGG C 3' lco384- pSKpjrev (22 mer) pBluescript reverse 1 5' CTA AAG GGA ACA AAA GCT GGA G 3' lco385- pSKrev (21 mer) pBluescript reverse2 5' CAG CTA TGA CCA TGA TTA CGC 3' Equipment list: Supplies List: Table 2 Variation of PBGD clones from published erythroid sequence: Table 2: Summary of the number of differences in amino acid sequence of our sequenced PBGD clones and clones from Genebank entrys for the constitutive and genomic PBGD with published Erythropoietic PBGD sequence. Shown in different columns are the total number of silent mutations with a DNA base change not causing a corresponding amino acid change, the number of non-silent mutations with a DNA change causing an amino acid difference and the sum of the two types of mutations. Not included in this figure are the three silent mutations introduced into the clones to create an internal Mun-I endonuclease cleavage site. Note that clone number 1.1 which will be used for production of recombinant human porphobilinogen deaminase (rh-PBGD) has only one of each type of difference with the least number of total differences. Table 3: Summary of the genetic differences of our sequenced PBGD clones and Genebank entrys for the constitutive and genomic PBGD with published erythropoietic PBGD sequence from the allele sequence alignment. Listed in different columns are the amino acid, base number from the ATG start codon, the actual genetic difference with corresponding amino acid change if any and a listing of the clones with differences shown with an X. The final column tallys the number of clones with that mutation. The final four mutations are introduced with lco376 during PCR amplification to create a Mun-I endonuclease cleavage site. Note that clone number 1 .1 which will be used for production of rh-PBGD only has differences which are represented by a number of clones. Expression plasmids The expression plasmid pExpO (see Fig. 2) was constructed by excising the PBGD coding sequence (nt1-1043) from pPBGD1.1 (see Fig. 3) with EcoR I and Hind III and inserting it into the vector pKK223-3 (see Fig. 4) (Pharmacia Cat # 27-4935, Lot # 8054935011) cut with the same enzymes, thus operatively linking it to the inducible tac promoter. Another plasmid, pExpl (see Fig. 5) was also constructed with modifications to the 5' untranslated region and the initial part of the coding sequence both aimed at improving translation efficiencies (translation enhanced). The changes are indicated below and include, insertion of a second ribosome binding site, an AT rich sequence preceding the ATG and three silent base substitutions shown in as underlined. AATTCTAACA TAAGTTAAGG AGGAAAAAAA A ATG AGA GTT ATT CGT GTC GGT AC Met Arg Val He Λrg Val Gly Plasmid pExpl (Fig. 5) was made in a two step process. Oligonucleotides ICO386 (5' AAT TCT AAC ATA AGT TAA GGA GGA AAA AAA AAT GAG AGT TAT TCG TGT CGG TAC 3') and ICO387 (5' CGA CAC GAA TAA CTC TCA TTT TTT TTT CCT CCT TAA CTT ATG TTA G 3') were designed to provide upon annealing a 5' EcoR I adhesive end and a 3' Kpn I sticky end. Oligonucleotides ICO386 and ICO387 were annealed and ligated with the Kpn \-Hind III PBGD fragment from pPBGDI .1 into EcoR I -Hind III linearized pBluescript II SK- (Stratagene Cat # 212206) to yield plasmid pPBGDI .1Tra (Fig. 6). In the second step the EcoR I -Hind III fragment from pPBGDI .1Tra was ligated into pKK223-3 cut with the same enzymes resulting in plasmid pExpl (Fig. 5). Host strains and growth conditions For the studies done so far two E. coli K12 strains were used. Both these strains carry the lacl q repressor and are, DH12S (Life Technologies cat # 1832-017) and JM105 (Parmacia Cat # 27-1550-01). The media used was mainly LB Broth (Difco Laboratories Cat # 0446- 07-5) containing 100 μg/ml ampicillin. The cultures were grown at 37° C and induced with 4mM IPTG for 3 hours. The choice of the final strain will partly depend on the induction strategy used (see below). The use of laclts will force the use of strains that do not carry the lacl q repressor. Further, strains deficient in proteases such as La, Clp and the rpoH locus may prove to be beneficial if thermal induction is to be used. The hemC gene is deleted in the final production strain. A plasmid, which will be used as a tool for this purpose has been constructed. Restoration of tetracycline resistance gene Plasmid pExp1-M2 (Fig. 7) has been constructed from pExpl with a functional tetracycline resistance gene. This was designed with a view to be able to introduce other regulatory elements or genes as required by the project as also being able to retain the ability to replace the "translation enhanced" PBGD sequence by the native sequence or a polylinker for the blank plasmid. The following strategy was used. Plasmid pExpl was cut with Sal I and Satr?H I and the 5348 base-pair fragment containing part of the tetracycline coding sequence and the bulk of the plasmid was isolated. Into this was ligated the Sal \-Hind III fragment from pBR322 (New England BioLabs Cat # 303-3S, Lot # 50) containing rest of the coding sequence and an adapter formed by annealing oligonucleotides ICO424 (5' GATCACTCAT GTTTGACAGC TTATCATCGA TT 3') and ICO425 (5. AGCTAATCGA TGATAAGCTG TCAAACATGA GT 3'). The adapter contains part of the tetracycline promoter and provides Hind III and Bam I overhangs for ligation but destroys the Hind III and BamH I restriction sites. Induction: The tac promoter is a strong promoter when induced with IPTG and other groups have shown that it or the other almost identical trc promoter can be induced very well at 42°C when a thermosensitive lac repressor is used. With pExp1-M2-Puc-BB construct a high level of expression which can translate to gram quantities of product in the fermenter. Based on study data and data from the literature the thermal induction is efficient. Strategy 1: The plasmid pExpl (see Fig. 5) has a basal level of expression which is about one-fifth that of a fully induced culture in a lacl q strain in LB broth. Stable and with acceptable expression levels, it can be used without induction in defined fermentation media. In the plasmid pExp1-M2 (see Fig. 7) the rom gene has been deleted on the plasmid and the pBR223 replicon exchanged by the pUC-based replicon. These plasmids have elevated copy numbers with increasing temperature (greater than 30°C) and serve as a pseudo-induction system. Strategy 2: In plasmid pExp1-M2 a thermo-labile la repressor has been introduced either at the Sea 1 site or the βsaA 1 site. The repressor was derived from plasmid pTYBI (New England BioLabs Cat # 6701 , Lot # 3) by PCR, wherein Gly 187 was changed to Ser. If the thermal induction works but a high basal level of expression leads to instability, the native PBGD sequence as in pExpO (without the enhancements) is substituted. In plasmid pExpO the uninduced level of expression is below the level of detection in the assay system used. Other variations would be to use lacl q ts on the plasmid or in the genome. Detailed Description of the Construction of the production plasmid for rPBGD The production plasmid pExp1-M2-Puc-BB (see Fig. 8) for rPBGD was constructed in a multi-step process. The individual steps used and all the intermediate plasmids are outlined below. The expression plasmid pExpl was first constructed wherein the PBGD coding sequence is operatively linked to the tac promoter of the vector pKK223-3 (Pharmacia Cat # 27-4935, Lot # 8054935011) by means of a short adapter designed to include a ribosome binding site, an AT-rich region preceding the ATG and the first six codons of the PBGD human erythropoietic enzyme with three silent base changes. The sequence of the adapter is shown below with the base changes introduced in the wobble positions boldfaced. AATTCTAACA TAAGTTAAGG AGGAAAAAAA A ATG AGA GTT ATT CGT GTC GGT AC Met Arg Val He Arg Val Gly Oligonucleotides ICO386 (5' AAT TCT AAC ATA AGT TAA GGA GGA AAA AAA AAT GAG AGT TAT TCG TGT CGG TAC 3') and ICO387 (5' CGA CAC GAA TAA CTC TCA TTT TTT TTT CCT CCT TAA CTT ATG TTA G 3') were designed to provide upon annealing a 5' EcoR I adhesive end and a 3' Kpn I sticky end. Oligonucleotides ICO386 and 1CO387 were annealed and ligated with the Kpn \-Hind III PBGD fragment from pPBGDI .1 into EcoR I -Hind III linearized pBluescript II SK- (Stratagene Cat # 212206) to yield plasmid pPBGDI .1Tra. In the second step the EcoR \ -Hind III fragment from pPBGDI .1Tra was ligated into pKK223-3 cut with the same enzymes resulting in plasmid pExpl . The teracycline resistance gene was next restored using the following strategy. Plasmid pExpl was cut with Sal I and SamH I and the 5348 base-pair fragment containing part of the tetracycline coding sequence and the bulk of the plasmid was isolated. Into this was ligated the Sal \-Hind III fragment from ρBR322 (New England BioLabs Cat # 303-3S, Lot # 50) containing rest of the coding sequence and an adapter formed by annealing oligonucleotides ICO424 (5' GATCACTCAT GTTTGACAGC TTATCATCGA TT 3') and ICO425 (5. AGCTAATCGA TGATAAGCTG TCAAACATGA GT 3'). The adapter contains part of the tetracycline promoter and provides Hind III and SamH I overhangs for ligation but destroys the Hind III and BamH I restriction sites. The resulting plasmid was called pExp1-M2. Plasmid pExp1-M2 was digested with Pvu I and Afl III and the larger of the two fragments corresponding to a size of 4745 base-pairs was isolated. This was ligated to the 1257 base-pairs long Pvu l-Afllll fragment derived from pUC19 containing the origin of replication and part of the ampicillin resistance gene to obtain plasmid pExp1-M2-Puc. This was passaged through JM110 and cut with BsaA1 and BsaB1 to excise the rom gene contained between the two sites and blunt-ended together to yield the final expression plasmid pExp1-M2-Puc-BB. The pExp1-M2-Puc-BB plasmid has been fully sequenced (see sequence 11). Expression of rPBGD in E. coli The E coli K12 host strain JM105 genotype endA thi rpsL sbcB15 hsdR4 Δ(lac-proAB) [F'traD36 proAB lacl q Δ(lacZ)M15 ] containing the expression plasmid pExp1-M2-Puc-BB was grown in LB broth containing 100 μg/ml ampicillin at to mid-log phase at 30°C from a 1 to 40 dilution of an overnight inoculum. The culture was then split into three and growth was continued for another 4 hours at 30°C, 37°C and 42°C respectively. Cells were spun down from 1 ml samples and frozen at -20°C. The thawed cell pellets were resuspended in 200-300 μl of B-PER reagent PIERCE Cat. # 78243, incubated at room temperature for 10 minutes, spun at 16,000 for 10 minutes and PBGD activity was determined in the supernatants. Total protein was estimated by the Bradford method using the BioRad reagent Cat # 500-0006 and bovine serum albumin as stsndard. The specific activities in the crude lysates obtained at the three growth temperatures are tabulated below. The results clearly show an increase of PBGD units/mg with increasing temperature in the range from 30°C to 40°C. Temperature PBGD Units/mg 30°C 363 37°C 573 42°C 1080 OTHER PRODUCTION SYSTEMS FOR rPBGD For yeast production, the PBGD coding sequence can be inserted into a plasmid vector, for example YEP type, containing 2 u circular DNA (Ori) origin for high expression in yeast. YEP plasmids contain TRP 1 and URA 3 as markers for selective maintenance in trpl -, ura 3-yeast strains. Alternatively, the PBGD gene can be inserted in bovine papilloma virus vectors BPV for high expression in a murine cell line C-1 27 (Stephens P.E. et. al. Biochem J. 248, 1 - 1 1 , 1 987) or vectors compatible with expression in CHO cells or COS cells. An expression of PBGD can be made intracellularly. A secretory signal in Saccharomyces, for example alpha-mating factor presequence, can be added in front of the rPBGD structural gene for efficient secretion in yeast. Similarly, a sequence encoding a mammalian signal peptide can be added for secretion of rPBGD into the culture medium upon expression in for example CHO cells or COS cells. A bacterial promoter for example the tryptophane (trp) promoter or the lac promoter or alternatively an alkaline phosphatase promoter, should be inserted before the PBGD coding region for efficient transcription in prokaryotes for example E. coli. A yeast promoter for example 3-phosphoglycerate kinase (PGK) or chelatin or alpha- mating factor should be inserted before the PBGD coding region for efficient transcription in yeast for example Saccharomyces cerevesiae or Saccharomyces pombe. A mammalian promoter for example Metallothionin-1 (MT-1 ) or Aspartate transcarbamylase or Dihydrofolate reductase (DHFR) should be inserted before the PBGD coding region for efficient transcription in mammalian cell lines for example CHO cells or COS cells. The yeast plasmid (Y-G&F-PBGD) containing a yeast promoter, signal and/or ATG codon (methionine) in front of the PBGD coding region and a yeast vector containing selectable markers such URA 3 or TRP 1 will be transformed into the yeast host cell such as Saccharomyces cerevesiae or Saccharomyces pombe for production of rPBGD. The mammalian plasmid (M-G&F-PBGD) containing a mammalian promoter for example Metallothionine-1 or Dihydrofolate reductase and a mammalian signal sequence or an ATG codon in front of the PBGD coding region and vector pAT or pSV2 respectively. Plasmid (M-G&F PBGD) will be transfected into a mammalian cell line for example CHO cells, for production of rPBGD. The E. coli cell containing plasmid (pExpl or pExp1 -M2 Puc-BB), will be fermented to stationary phase between 24-48 hours, in a medium containing casein hydrolysate, or yeast extract, glucose, vitamins and salts. pH oxygen will be monitored by electrodes during fermentation. Temperature will be kept at 37 + /- 2 C during the fermentation. The yeast cell containing the plasmid (Y-G&F-PBGD), will be fermented to late log phase between 20-40 hours in a medium containing yeast extract, glucose, salts and vitamins. pH and temperature will be monitored by specific electrodes during fermentation. Temperature will be kept at 30 + /- 2 C during fermentation. The mammalian cell line containing the plasmid (M-G&F-PBGD) will be fermented in a medium containing, foetal calf serum (or serum free), vitamins, glucose, antibiotics, growth factors. pH and temperature will be monitored continuously during fermentation by specific electrodes. Fermentation and Purification rPBGD will recovered from E. coli after fermentation by an extraction procedure involving for example ribipress, sonication, osmotic shock or total solubilization by detergent for example Tween 80, Triton X-1 00 or Brij. rPBGD will be recovered from fermentation medium after production in yeast or from a total cellular extract using detergents such as Triton X-1 00, Tween 80 or Brij. rPBGD will be recovered from mammalian culture medium or from a total cellular extract by ion-exchange chromatography or affinity chromatography. rPBGD will be purified from E. coli extract or from yeast medium or total cellular extract or from mammalian culture medium or total mammalian cellular extract by binding to an ion-exchange column for example DEAE-Sepharose or MonoQ-Sepharose and eluted with for example NaCI and Sodium phosphate buffer pH 7-8 or the corresponding potassium salts. Alternatively, rPBGD will be recovered from extracts by binding to an affinity chromatography column for example an anti-PBGD affinity column. rPBGD will be eluted by lowering the pH to 4-2, or a thiol specific affinity column. rPBGD has been "tagged" with thiols residues when a thiol affinity column step is used. Thiols will be removed by a specific enzymatic cleavage step to generate authentic rPBGD. The ion-exchange or affinity purified rPBGD will be further purified by hydrophobic interaction chromatography on for example, TSK Phenyl 5 PW column or Octyl- Sepharose or Phenyl-Sepharose columns. Binding of rPBGD will be done at high ionic strength for example in 10-50 mM Tris- HCI pH 7-8, 1 M NaCI or 10-1 5 mM Sodium phosphate pH 7-8, 0.5 M MgS0 4 and eluted by lowering the ionic strength for example with 10-50 mM Tris-HCI pH 7-8 or 10-50 mM Sodium phosphate pH 7-8. Three hydrophobic interaction steps will be applied consecutively. rPBGD will be further purified with preparative RP-HPLC for example C1 2 or C18 matrixes. The rPBGD will be eluted from the column by a gradient of 10-50 mM Sodium phosphate and 1 -10% acetonitrile buffer. Formulation of rPBGD will be done by passing the enzyme over a G-1 00 Sephadex column and eluting it in an isotonic solution for example 0.9%NaCI and 10-50mM Sodium phosphate pH7.0 + /- 0.5 or Sodium phosphate, glycin, mannitol or the corresponding potassium salts. The formulation solution of rPBGD will be sterile filtered and filled aseptically in glass vials and lyophilised. Alternatively, the sterile filtered rPBGD solution will be formulated in for example, lipid vesicles constituting phosphatidylcholine or phosphatidylethanolamine or combinations of these or incorporated into erythrocyte ghosts. Reconstitution of lyophilised rPBGD will be done in water for injection. Alternatively, rPBGD will be formulated in a sustained release formulation involving a biodegradable microspheres, for example in polylactic acid, polyglycolic acid or mixtures of these. Alternatively, rPBGD will be lyophilized in a two-compartment cartridge, where rPBGD will be in the front compartment and water for reconstitution in the rear compartment. This two compartment cartridge will be combined with an injection device to administer either rPBGD by a needle or needle less (by high pressure) device. Alternatively, rPBGD will be formulated in a physiological buffer containing an enhancer for nasal administration. Alternatively, rPBGD will be formulated in an oral formulation containing for example, lipid vesicles (phospatidylcholine, phosphatidylethanolamine, sphingomyeline) or dextrane microspheres. Although recombinant production of PBGD is preferred for the treatment of AIP, it can alternatively be produced from human red blood cells. The production and manufacturing of recombinant PBGD will be done by the following steps. Recombinant PBGD production process; an outline A: Fermentation 1 . Master cell bank 2. Working cell bank 3. Production of seed culture 4. Fermentation in large fermenter (250 L > ) B. Purification 1 . Cell concentration by filtration/centrifugation 2. Cell disruption 3. Ultrafiltration 4. Chromatography ion exchange, DEAE-Sepharose, MonoQ-Sepharose 5. Hydrophobic interaction chromatography (Octyl/phenyl-Sepharose, TSK Phenyl, 5PW, Phenyl -Sepharose 6. Chromatography ion exchange (MonoQ) 7. Formulation by Gel filtration Sephadex G-100 C. Manufacturing 1 . Sterile filtration 2. Aseptic filling 3. Lyophilization TREATMENT OF OTHER PORPHYRIAS In analogy with the new treatment of AIP patients with (recombinant) PBGD, hepatic Porphyrias such as ALA deficiency Porphyria (ADP), Porphyria cutanea tarda (PCT), Hereditary Coproporphyria (HCP) and Variegata Porphyria (VP) can benefit from substitution therapy by rALA dehydratase, rUroporphyrinogen decarboxylase, rCoproporphyrinogen oxidase and rProtoporphyrinogen oxidase, respectively. Patients having Erythropoetic Porphyrias such as Congenital erythropoietic Porphyria (CEP) or Erythropoietic protoporphyria (EPP) will benefit from substitution therapy with rUroporphyrinogen III syntetase and rFerrochelatase, respectively. Hepatoerythropoietic Porphyrias e.g. Hepatoerythropoietic Porphyrias(HEP) can be treated with rUroporphyrinogen decarboxylase. All porphyrias can be treated by the administration of the enzymatic activity lacking or being reduced (normally 50%) in any of the eight steps in the heme biosynthetic pathway as described above. The substitution of the enzymatic activity can be achieved by adding the corresponding recombinant enzyme or other molecules that will provide the missing enzymatic activity. GENE THERAPY AS AN ALTERNATIVE TREATMENT POSSSIBILTY FOR PATIENTS WITH ACUTE INTERMITTENT PORPHYRIA (AIP) The human enzyme Porphobilinogen deaminase PBGD is coded for by a single gene located on chromosome 11q 24. Mutations in this gene causes the disease Acute Intermittent Porphyria (AIP). The disease has been shown to be inhereted in an autosomal dominat way. Today over 100 mutations in the PBGD gene has been identified (Grandchamp B. J. Ganstroenterology and Hepathology, 11 , 1046-1052, 1996, Table A) and the number is expected to increase when modern diagnostic systems based on screening programs will be applied more routinely in hospitals. A number of these mutations are shown in Table A Table A Reported mutations in the PBGD gene Position Mutation Consequences Reference Exon 1 3 ATG→ATA Translation impairment 18 33 GCG→GCT DS 17 Intron 1 33 + 1 gtg→atg DS 16 Exon 3 76 CGC→TGC R26C 25 77 CGC→CAC R26H 26 Exon 4 91 GCT→CACT A31 T 24 97 Del A Frameshift 25 100 CAG→AAG Q34K 27 100 CAG→TAG Q34X 25 125 TTG→TAG L42X 19 Exon 5 163 GCT→TCT A55S 24 174 Del C Frameshift 24 182 Ins G Frameshift 24 Intron 5 210 + 1 gta→ata DS (Del exon 5) 24 Exon 6 218-219 Del AG Frameshift 24 Exon 7 277 GTT→TTT V93F 24 293 AAG→AGG K98R 25 331 GGA→AGA G 1 1 1 R 28 Intron 7 345-1 cag→caa AS (Del exon 8) 29 Exon 8 346 CGG-→TGG R1 16W 20 347 CGG→CAG R1 16Q 30 Exon 9 445 CGA→TGA R149X 25 446 CGA→CAA R149Q 31 446 CGA→CTA R149L 24 463 CAG→TAG Q1 55X 32 470 Ins A Frameshift 29 Table A cont. Position Mutation Consequences References Intron 9 499-1 cag→caa AS (Del exon 10) 21 Exon 10 499 CGG→TGG R167W 33 500 CGG→CAG R167Q 27,34 518 CGG→CAG R173Q 34 530 CTG→CGG L177R 27 593 TGG→TAG W198X 19 604 Del G Frameshift 35 610 CAG→TAG Q204X 30 612 CAG→CAT DS (Del 9 bp exon 10) 31 Exon 1 1 625 GAG→AAG E209K 28 Intron 1 1 652-3 cag→gag AS (Del exon 12) 33 Exon 12 667 GAA→AAA E223K 24 673 CGA→GGA R225G 25 673 CGA→TGA R225X 25 713 CTG→CGG L238R 25 715-716 Del CA Frameshift 19 730-731 Del CT Frameshift 36 734 CTT→CGT L245R 31 739 TGC→CGC C247R 36 740 TGC→TTC C247F 18 742 Ins 8 bp Frameshift 24 748 GAA→AAA E250K 24 754 GCC→ACC A252T 36 755 GCC→GTC A252V 36 766 CAC→AAC H256N 27 771 CTG→CTA DS (Del exon 12) 39 771 CTG→CTC DS (Del exon 12) 37 Intron 12 771 + 1 gta→ata DS (Del exon 12) 19 Table A cont. Position Mutation Consequences References Exon 13 806 ACA→ATA T269I 30 820 GGG→AGG G274R 30 Exon 14 838 GGA→AGA G280R 25 848 TGG→TAG W283X 30 886 CAG→TAG Q296X 25 900 Del T Frameshift 31 Intron 14 912 + 1 gta→ata DS (Del exon 14) 28 Exon 15 1062 Ins C Frameshift 38 1073 Del A Frameshift 25 In one aspect, the present invention relates to a therapeutic method for AIP patients based on gene therapy. The gene therapy treatment may involve the following steps. 1. Identification mutations in the PBGD gene causing AIP in humans 2. Selection of human PBGD cDNA sequence for gene therapy 3. Construction of PBGD gene therapy vectors. 4. Production of PBGD gene transfer vector 5. Delivery system of PBGD gene transfer vector 1. Identification of mutations in the PBGD gene causing AIP in humans Patients having a point mutation in Exon 10 at position 593 TGG>TAG have a change in the amino acid sequence of the PBGD enzyme from W198X (stop codon). This mutation is carried by approximately 50 % of all AIP patients in Sweden (Lee JS. et al. Proc. Natl. Acad. Sci. USA, 88, 10912-10915, and 1991). AIP patients with other mutations than W198X, which might also benefit from gene therapy, are given in Table A. 2. Selection of human PBGD cDNA sequence for gene therapy There are two isoenzyme forms of human PBGD e.g. erythropoietic and the non- erythropoietic form, which are formed by an alternative splicing mechanism. The non-erythropoietic form has a 17 amino acid extension on the N-terminal end of the erythropoietic PBGD form. Non-erythropoietic PBGD form (nPBGD): Met-Ser-Gly-Asn-Gly-Asn-Ala-Ala-Ala-Thr-Ala-Glu-Glu-Asn-Ser-Pro-Lys-Met-Arg-Val.... ATG-TCT-GGT-AAC-GGC-ATT-GCG-GCT-GCA-ACG-GCG-GAA-GAA-AAC-AGC-CCA-AAG-ATG- AGA-GTG.. Erythropoietic PBGD form (ePBGD): Met-Arg-Val- ATG-AGA-GTG... The nucleotide and amino acid sequence for human PBGD that will be used for gene therapy differs from that published by Raich N. et al. Nucl. Acid. Res. 14, 5955-5968, 1986 in that the amino acid residue in position 332 is an Asn residue rather than Thr. In order to make the "wild type enzyme" and avoiding formation of antibodies the PBGD sequence has to contain an Asn residue in position 332. The cDNA sequence that will be used for the erythropoietic PBGD form is shown above. Patient with a defect erythropoietic PBGD enzyme will be transfected with the erythropoietic PBGD cDNA sequence and patients with a defect in the non-erythropoietic form will be transfected with the non-erythropoietic cDNA sequence. 3. Construction of PBGD gene therapy vectors Adenoviral vector system The vector is based on adenovirus type 5 (Ad5), containing three essential genetic loci E.g. E1 , E2, E4, encoding important regulatory proteins and one locus E3 which is non- essential for virus growth. Deletion of E1A and E1 B region renders the virus replication deficient in vivo. Efficient complementation of the E1 function (recombinant viral stocks) can be obtained in an E1 expressing cell line such as human 293-cell line. The human PBGD cDNA will be inserted in an adenovirus vector system. The PBGD transgenes will be driven by the endogenous PBGD promoter or a cytolomega virus promoter (CMV). Retroviral vectors Retroviral vectors are well suited for gene delivery for several reasons: 1. simplicity 2. capacity to integrate up to 8kbp DNA inserts 3. their safety, non pathogenic to humans 4. easy to improve and manipulate 5. defined integration sites of genes 6. long term regulated expression One major disadvantage with the retroviral vectors though, is that they can only transduce dividing cells. Most common retroviridae considered for gene therapy, are the lentiviridae and the mammalian C-type viridae. Other type retroviruses have also been considered. One such example, is a Moloney-murine leukemia retrovirus (Mo-MLV), which has been successfully used to transduce mouse and human fibroblasts with the uroporphyrinogen III synthetase (UROIIIS). (Moreau-Gaudry et al. Human Gene Therapy 6, 13-20,1995). The expression of the UROIIIS gene was driven by long terminal repeat (LTR). The UROIIIS cDNA was also successfully transduced by the retrovirus vectors into human peripheral blood progenitor cells. The erythropoietic PBGD cDNA sequence can be inserted in a retrovirus vector LXSN (Miller et al BioTechniques 7, 980-990, 1989) and pMFG ( Dranoff et al. Proc. Natl. Acad. Sci. USA. 90,3539-3543, 1993). This will lead to the following constructs e.g. LePSN and pMFG-ePBGD, respectively. LePSN: > 1032 bp < LTR / cDNA ePBGD / SV40 / Neo / LTR pMFG-ePBGD: > 1032 bp < LTR / cDNA ePBGD/ LTR For transduction of non-erythropoietic tissues the non-erythropoietic cDNA (See sequence 12) will be inserted in the LSXN vector and the pMFG vector resulting in the LSnPN and pMFG-nPBGD vectors, respectively. LnPSN: > 1083 bp < LTR / cDNA nPBGD / SV40 / Neo / LTR pMFG-nPBGD: > 1083 bp < LTR -/ cDNAnPBGD/ LTR The LePSN and LnPSN vectors can be converted to the corresponding virus by transfer into an appropriate host cell line e.g. Ψ CRE as described by (Danos et al. Proc. Natl. Acad. Sci. USA. 85, 6460-6464, 1988). Filtered supernatants from ectopic virus producing cells were added to amphotropic cells Ψ CRIP, in the presence of Polybrene. Clones can be isolated and tested for virus. Clones that show titers over 1.000.000 cfu/ml will be saved (resistant to G418). The LnPSN vector will be cotransfected with the pMCI-Neo plasmid (Pharmacia, Sweden) into the packaging cell line Ψ CRIP. Clones that shows integration of provirus and high expression levels of message will be selected. Filtrate from supernatants from virus producing cells (erythropoietic PBGD form) can be mixed with Polybrene and incubated with peripheral blood progenitor cells (bone marrow transplant) from an AIP patient for several hours. The transduced progenitor cells can then be transplanted back into an AIP patient. The success of the treatment will be measured as the increase in the PBGD activity in erythrocytes and reduced excretion of ALA and PBG in the urine. Clinically a success of the treatment can be evaluated as a reduction of frequency of spontaneous acute attacks or drug-induced attacks. This will be a more convenient way of administering the recombinant PBGD enzyme than regular injections. The efficacy of the therapy can be evaluated by measuring the PBGD activity in blood and reduced excretion of PBG and ALA in the urine. Clinically, a successful treatment should result in less number of acute attacks or preferably no more attacks. Associated Adenovirus system (AAV) AAV is a non-pathogenic human virus (Parvovirus) carried by more than 80% of all people. The advantage with AAV as compared to retroviral systems is that AAV can transduce both dividing and non-dividing cells. The virus genome, which is small, contains two Inverted Therminal Repeats (ITR) and a REP and CAP functions. The REP and CAP functions can be deleted and exogenous cDNA inserted. Construction of an AAV vector containing the erythropoietic PBGD cDNA can be made. This AAV/PBGD vector will be suitable to transduce AIP patient's bone muscle cells, as a " muscle factory" for PBGD enzyme production. The PBGD cDNA will be engineered in such a way that a signal sequence for secretion will be added on the 5 ' -end of the cDNA. This will allow the erythropoietic PBGD enzyme to become secreted from the muscle cells into the blood stream. By this system patients will receive a constant delivery of active PBGD enzyme into the bloodstream, which will metabolize PBG thereby avoiding acute attacks. - Non-erythropoietic Alternatively, liver cells can be transduced with AAV containing the non-erythropoietic PBGD cDNA. The construct will be engineered in such a way that the translated PBGD enzyme will remain intracellular e.g. contain a Met residue at the N-terminal end of the PBGD enzyme without a signal sequence for secretion in mammalian cells. The PBGD transgene will be transcribed and translated into new PBGD enzymes that will remain intracellularly. Levels of new PBGD enzymes made in the liver will be normalized the PBGD activity to 100%. AIP patients have usually reduced PBGD activity (50-80%) in the liver depending on the mutation and individual variations. This treatment would aliviate the clinical symptom e.g. acute attacks with abdominal pain and reduce excretion of PBG and ALA in the urine. The AAV containing the non- erythropoietic PBGD form can also be used to correct the genetic defect in other cell types such as neuronal tissue, pancreas spleen e.g. non-erythropoietic tissue, by a similar mechanism. - Erythropoietic The erythropoietic PBGD cDNA can be inserted in an AAV vector and used to transduce erythropoietic cells and stem cells in AIP patients, having a mutation affecting the erythropoietic form of PBGD. 4. Production of PBGD gene transfer vector Adenovirus have approximately 36 kbp double stranded DNA, containing three essential early gene loci (E1 , E2, and E4) encoding important regulatory proteins. Loci E3 codes for a gene product that block immune response to virus infected cells in vivo. The PBGD gene transfer adenovirus vector can be produced by deleting the E1 and E3 loci. The PBGD gene cassette is inserted in that position instead. The virus will be replication defective when the E1 locus has been deleted. Efficient E1 complementation and thus high yield of recombinant virus vector (PBGD) can be obtained in an E1 expressing cell line, such as the human 293 cell line. (Graham, F. et al. 1977, Characteristics of a human cell line transformed by DNA from human adenovirus 5. J. Gen. Virol. 36, 59-72). 5. Delivery systems of PBGD gene transfer vectors. Delivery of viral vectors are based on injection into the patient of a virus particle that will transduce human cells in vivo. Correction of point mutations causing AIP by Chimeraplasty Gene Repair The basic technique involves the synthesis of chimeric (RNA-DNA) oligonucleotides. The oligonucleotide will repair point mutations on the chromosome by binding to the site of mutation and create a mismatch. The endogenous "mismatch repair system" which is present in all living cells, will correct the mutation. The Chimeric oligonucleotides has the following general properties: a. 68 mer (65-70 is acceptable size) b. 25 base DNA stretches at the 5 ' -end homologous to the normal sequence of the gene c. the 25 base DNA is designed in such a way the 12 bp on each side of the mutation is complementary to "wild-type DNA" where the mutation to be altered is located at position 13 d. the 25 mere contains 4 T bases at the one end to loop back the oligo to the other DNA strand with a 25 base sequence homologous to the other strand of the chromosomal DNA. e. the second strand is chimeric in that it contains 10 homologous bases of 2O methyl RNA followed by 5 bases of DNA (containing a central mismatch e.g. correction of the human point mutation by mismatch repair) followed by another stretch of 10 bases of homologous 2O methyl RNA. This stretch of DNA/RNA is followed with 5 bases of GC clamp and 4 T bases to form the second loop and finally a 5 base CG clamps complementary to the other one. EXAMPLE 1 Correction of the PBGD mutation at position 593 TGG>TAG resulting in W198X Normal Chromosomal Sequence: 5 ' -AG CGC ATG GGC TGG CAC AAC CGG GT-3 ' Gin Arg Met Gly Trp His Asn Arg Val AIP Chromosomal Sequence: 5 ' - AG CGC ATG GGC TAG CAC AAC CGG GT-3 ' Stop The sequence of the chimeric oligonucleotide ( Heme593W/X) is: DNA G RNA i i RNA Chromosome 5 ' - AG CGC ATG GGC TAG CAC AAC CGG GT-3 ' . gggccTC GCG TAC CCG ACC GTG TTG GCC CA T T ( loop T Heme593W/X T loop T T I ) Tcccgg T 5 ' -AG CGC ATG GGC TGG CAC AAC CGG GT Chromosome .... 3 ' -TC GCG TAC CCG ATC GTG TTG GCC CA -5'... rt C The same principle of chimeric oligonucleotide can be constructed to correct any of the mutations causing AIP depicted in Table A. Chimeric oligonucleotides can be used to correct any other point mutation causing any of the 8 known Porphyrias in a similarly as described above. Delivery of PBGD gene transfer of non viral vectors to humans The chimeric oligonucleotide can be formulated in a vechicles preparation containing anionic or cationic phospholipids or phospholipids mixed with neutral lipids or lictosylated PEL Alternatively, the non-viral vectors can be formulated in liposomes containing mixtures of natural phospholipids and neutral lipids. Specific protein seguences can be incorporated into liposomal membranes, that recognizes cellular receptors for specific targeting of non-viral vectors to a specific cell type such as liver, neuronal tissue or erythropoietic tissues, can be incorporated. Alternatively specific antibodies recognizing specific cellular surface antigens can be used for targeting. Thirdly, carbohydrates on the liposomal membrane can be used for liver uptake of chimeric oligonucleotides. The formulated chimeric oligonucleotide (HemeBiotech 595 W/X) will be administered by sc. or IV. injections to AIP patients. The efficacy of the treatment can be evaluated as above. GENE THERAPY AS AN ALTERNATIVE TREATMENT OF OTHER PORPHYRIC DISEASES The gene therapy strategies outlined herein can also be used for other Porphric diseases. The general principle is to increase the cellular or systemic content of a particular defective enzyme causing the disease. The following Porphyric diseases can be encompassed by this strategy: 1. ALA deficiency porphyria (ADP) 2. Porphyria cutanea tarda (PCT) 3. Hereditary coproporphyria (HCP) 4. Harderoporphyria (HDP) 5. Variegata porphyria (VP) 6. Congenital erythropoietic porphyria (CEP) 7. Erythropoietic protoporphyria (EPP) 8. Hepatoerythropoietic porphyria (HEP) References: Andersson, Christer, Thesis, 1997, ISBN 91 /71 91 /280/0, pp. 22-23 Danos et al. Proc. Natl. Acad. Sci. USA. 85, 6460-6464, 1988 Dranoff et al. Proc. Natl. Acad.Sci. USA. 90,3539-3543, 1993 Grandchamp B, et al Tissue-specific expression of porphobilinogen deaminase. Two isoenzymes from a single gene. Eur J Biochem. 1 987 Jan; 162(1 ): 105-10. Grandschamp B. et al. 1996. J. of Gastroenerology and Hepatology 1 1 , 1046-1052 Herrick A.L. et al 1989, Lancet 1 , 1 295-1 297 Jeans J. et al. American J. of Medical Genetics 1 996,65, 269-273 Lithner F. et al. 1984, Acta.Med. Scand. 215,271-274 Maniatis T., E.F. Fritsch, J. Sambrook. Molecular Cloning (A laboratory Manual) Cold Spring Harbor Laboratory. 1982. Miller et al BioTechniques 7, 980-990, 1989 Moreau-Gaudry et al. Human Gene Therapy 6, 13-20,1995). Mustajoki et al. 1989, Sem. Hematol. 26, 1 -9). Raich N, et al Molecular cloning and complete primary sequence of erythrocyte porphobilinogen deaminase. Nucleic Acids Research 1 986 14(1 5): 5955-67 Raich N. et al 1986, Nucleic. Acid. Res, 14, 5955-5968 Saiki RK, et al Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985 Dec 20;230(4732):1350-4. Sassa S.1996, Blood Review, 10, 53-58 Stephens P.E. et. al. Biochem J.248, 1-11, 1987 Strand et al. Proc. Natl. Acad. Sci. 1970, 67, 1315-1320 Tishler P.V: et al. 1985, Am.J. Psychiatry 142,1430-1436 Waldenstrδm J. Acta.Med. Scand.1937 Suppl.82 Welland F.H. et al.1964, Metabolism, 13, 232 Wetterberg L.1967, Svenska bokfδrlaget Nordstedt, Stockholm Wetterberg L.1976, In Doss M. Nowrocki P. eds. Porphyrias in Human Disease. Reports of the discussion. Matgurg an der Lahn, 191-202 Yeung L. et al.1983, Q J. Med 52, 92-98 Yoo HW, et. Al. Hydroxymethylbilane synthase: complete genomic sequence and amplifiable polymorphisms in the human gene. Genomics 1993 Jan: 15(1 ):21-9.

Claims: CLAIMS 1 . A method for treatment or prophylaxis of disease caused by deficiency, in a subject, of an enzyme belonging to the heme biosynthetic pathway, the method comprising administering, to the subject, an effective amount of a catalyst which is said enzyme or an enzymatically equivalent part or analogue thereof. 2. A method according to claim 1 , wherein the disease is selected from the group consisting of acute intermittent porphyria (AIP), ALA deficiency porphyria (ADP), Porphyria cutanea tarda (PCT), Hereditary coproporphyria (HCP), Harderoporphyria (HDP), Variegata porphyria (VP), Congenital erythropoetic porphyria (CEP), Erythropoietic protoporphyria (EPP), and Hepatoerythropoietic porphyria (HEP). 3. A method according to claim 1 or 2, wherein the catalyst is an enzyme selected from the group consisting of porphobilinogen deaminase (PBGD) ALA dehydratase, Uroporphyrinogendecarboxylase, Coproporphyrinogen oxidase, Coproporphyrinogen oxidise, Protoporphyrinogen oxidase, Uroporphyrinogen III synthase, Ferrochelatase, and Uroporphyrinogen decarboxylase, or an enzymatically equivalent part or analogue thereof. 4. A method according to any of the preceding claims, wherein the disease is AIP and the enzyme is PBGD or an enzymatically equivalent part or analogue thereof. 5. A method according to any of the preceding claims, wherein the catalyst is a recombinant form of the enzyme belonging to the heme biosynthetic pathway or of the enzymatically equivalent part or analogue thereof. 5 6. A method according to any of the preceding claims, wherein the catalyst is administered by a route selected from the group consisting of the intravenous route, the intraarterial route, the intracutaneous route, the subcutaneous route, the oral route, the buccal route, the intramuscular route, the anal route, the transdermic route, the intradermal route, and the intratechal route. 10 7. A method according to any of claims 1 -6, wherein the catalyst is formulated in an isotonic solution, such as 0.9% NaCI and 10-50 mM Sodium phosphate pH 6.50 to 8 or Sodium phosphate, glycine, mannitoi or the corresponding potassium salts. 15 8. A method according to any of claims 1 -7, wherein the catalyst is lyophilised. 9. A method according to any of claims 1 -8, wherein the catalyst is sterile filtered. 10. A method according to any of claims 1 -6, 8 or 9, wherein the catalyst is 20 formulated as lipid vesicles comprising phosphatidylcholine or phosphatidylethanolamine or combinations thereof. 1 1 . A method according to any of claims 1 -6, 8 or 9, wherein the catalyst is incorporated into erythrocyte ghosts. 25 1 2. A method according to any of claims 1 -6, 8 or 9, wherein the catalyst is formulated as a sustained release formulation involving biodegradable microspheres, such as microspheres comprising polylactic acid, polyglycolic acid or mixtures of these. 30 1 3. A method according to any of claims 1 -9, wherein the catalyst is lyophilized in a two-compartment cartridge, where the catalyst will be in the front compartment and water for reconstitution in the rear compartment. 14. A method according to claim 1 3, wherein the two compartment cartridge is combined with an injection device to administer the catalyst either by a needle or by a needle-less (high pressure) device. 1 5. A method according to any of claims 1 -9, wherein the catalyst is formulated in a physiological buffer containing an enhancer for nasal administration. 16. A method according to any of the preceding claims, wherein the catalyst is formulated as an oral formulation containing lipid vesicles, such as those comprising phospatidylcholine, phosphatidylethanolamine, or sphingomyeline, or dextrane microspheres. 17. A method according to any of the preceding claims, wherein the catalyst is formulated so as to enhance the half-life thereof in the subject's bloodstream. 18. A method according to claim 17, wherein the catalyst has a polyethylene glycol coating. 19. A method according to claim 1 7, wherein the catalyst is complexed with a heavy metal. 20. A method according to any of the preceding claims, wherein the catalyst is an enzymatically equivalent part or analogue of the enzyme and exerts at least part of its enzymatic activity intracellularly upon administration to the subject. 21 . A method according to claim 20, wherein the catalyst is a small artificial enzyme or an organic catalyst which can polymerize porphobilinogen to hydroxymethylbilane 22. A method according to any of claims 1 -9, wherein the catalyst is said enzyme formulated in such a manner that it exerts at least part of its enzymatic activity intracellularly upon administration to the subject. 23. A method according to claim 22, wherein the catalyst is tagged with specific carbohydrates or other liver cell specific structures for specific liver uptake. 24. A method according to claims 1 -1 9, wherein the catalyst exerts substantially all its enzymatic activity extracellularly in the bloodstream. 25. A method according to claim 24, wherein the enzymatic activity of the catalyst on 5 its relevant heme precursor results in a metabolic product which 1 ) either moves into the intracellular compartment and is converted further via the remaining steps of the heme biosynthetic pathway or 2) is excreted from the subject via urine and/or faeces. 26. A method according to any of the preceding claims, wherein the catalyst has been 10 prepared by a method comprising a) introducing, into a suitable vector, a nucleic acid fragment which includes a nucleic acid sequence encoding the catalyst; b) transforming a compatible host cell with the vector; 15 c) culturing the transformed host cell under conditions facilitating expression of the nucleic acid sequence; and d) recovering the expression product from the culture and optionally subjecting the expression product to post-translational processing, such as in vitro protein refolding, enzymatic removal of fusion partners, alkylation of amino 20 acid residues, and deglycosylation, so as to obtain the catalyst. 27. A method according to any of claims 1 -25, wherein the catalyst has been prepared by liquid-phase or solid-phase peptide synthesis. 25 28. A method according to any of the preceding claims, wherein the catalyst is free from any other biological material of human origin. 29. A method according to any of the preceding claims, wherein the catalyst is administered at least once a day, such as 2, 3, 4, and 5 times daily. 30 30. A method according to any of the preceding claims wherein the daily dosage is in the range of 0.01 - 1 .0 mg/kg body weight per day, such as in the range of 0.05 - 0.5 mg/kg body weight per day. 31 . A method according to any of the preceding claims, wherein the daily dosage is about 0.1 mg per kg body weight per day. 32. A catalyst which is an enzyme of the heme biosynthetic pathway or an 5 enzymatically equivalent part or analogue thereof, for use as a medicament. 33. Use of a catalyst which is an enzyme of the heme biosynthetic pathway or an enzymatically equivalent part or analogue thereof for the preparation of a pharmaceutical composition for the treatment or prophylaxis of diseases caused by 10 deficiency of said enzyme. 34. The use according to claim 33, wherein the treatment or prophylaxis is performed according to any of claims 1 -31 . 15 35. A method according to any of claims 1 -31 wherein the catalyst is a recombinant form of the enzyme. 36. A method according to any of claims 1 -31 wherein the catalyst is recombinant human PBGD based on any of Seq. ID NO 1 (clone PBGD 1 .1 ) and Seq. ID NO 1 2 20 (non-erythro PBG D 1 .1 .1 ). 36. A catalyst according to claim 32, which is recombinant human PBGD based on any of Seq. ID NO 1 (clone PBGD 1 .1 ) and Seq. ID NO 1 2 (non-erythro PBGD 1 .1 .1 ). 25 37. A method for treating a patient having a mutation in the PBGD gene causing an enzyme defect, comprising the use of a human PBGD cDNA sequence of either non- erythropoietic form or erythropoietic form according to the tissue in which PBGD should be expressed, and transfection of the patient with the relevant cDNA. 30 38. The method according to claim 37 wherein the enzyme defiency is selected from enzyme defiencies resulting in a disease selected from Acute Intermittent Porphyria, (AIP), ALA deficiency porphyria (ADP), Porphyria cutanea tarda (PCT), Hereditary coproporphyria (HCP), Harderoporphyria (HDP), Variegata porphyria (VP), Congenital erythropoietic porphyria (CEP), Erythropoietic protoporphyria (EPP), and Hepatoerythropoietic porphyria (HEP). 39. The method according to claim 39 wherein the disease is Acute Intermittent Porphyria, (AIP). 40. The method according to any of claims 37-39 wherein the human PBGD cDNA sequence is selected from Seq. ID NO 1 (clone PBGD 1 .1 ) and Seq. ID NO 12 (non- erythro PBGD 1 .1 .1 ) 41 . The method according to any of claims 37-40 wherein the transfection is by use of a vector selected from adenovirus, retrovirus and associated adenovirus. 42. The method according to any of claims 37-41 wherein the PBGD gene transfer vector into human cells (erythropoeitic and/or non-erythropoietic) results in normal PBGD activity. 43. A method of gene therapy treatment of patients with Acute Intermittent Porphyria (AIP) by a correction of one of the specific point mutations identified causing AIP by use of chimeraplasty gene repair. 44. The method according to any of claims 43 wherein the delivery system for transfection is by use of non-viral vectors formulated in a vehicle preparation comprisng one or more components selected from cationic phospholipids, phospholipids, phospholipids mixed with neutral lipids, lictosylated PEI, liposomes liposomes comprising mixtures of natural phopholipids and neutral lipids. 45. A method according to claim 43 or 44 wherein the mutation is selected from Table A.

Inventors: FOGH JENS, GELLERFORS PAER, FOGH, JENS, GELLERFORS, PAER
Applicants: FOGH JENS, GELLERFORS PAER, HEMEBIOTECH A S, FOGH, JENS, GELLERFORS, PAER, HEMEBIOTECH A/S
Application year: 1999
Main classification: A61K38
IPC codes: A61K9, A61K38, A61K48, A61P7, A61P43, C12N9, C12N15