U.S. patent application number 10/393269 was filed with the patent office on 2003-12-04 for therapeutic method for treating patients with acute intermittent porphyria (aip) and other porphyric diseases.
Invention is credited to Fogh, Jens, Gellerfors, Par.
Application Number | 20030223979 10/393269 |
Document ID | / |
Family ID | 26788678 |
Filed Date | 2003-12-04 |
United States Patent
Application |
20030223979 |
Kind Code |
A1 |
Gellerfors, Par ; et
al. |
December 4, 2003 |
Therapeutic method for treating patients with 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 erythropoetic porphyria
(CEP), Erythropoietic protoporphyria (EPP), and
Hepatoerythropoietic porphyria (HEP). The catalyst is one or more
enzymes selected from the group consisting of
delta-aminolevulininic acid synthetase, delta-aminolevulinic acid
dehydratase (ALAD), porphobilinogen deaminase (PBGD),
uroporphyrinogen III cosythetase, uroporphyrinogen decarboxylase,
coproporphyrinogen oxidase, protoporphyrinogen oxidase, and
ferrochelatase, or an enzymatically equivalent part or analogue
thereof. In addition the invention relates to the use of PBGD, to
human recombinant PBGD and to a method of gene therapy. The
invention also relates to an expression plasmid pExp1-M2-BB (Seq.
ID No. 1) and to use of a DNA fragment, the EcoR I-Hind III linear
fragment (seq. ID No. 2), used for transformation in the hemC
disruption strategy for production of rhPBGD expressed in E.
coli.
Inventors: |
Gellerfors, Par; (Lidingo,
SE) ; Fogh, Jens; (Lynge, DK) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.
624 Ninth Street, N.W.
Washington
DC
20001
US
|
Family ID: |
26788678 |
Appl. No.: |
10/393269 |
Filed: |
March 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10393269 |
Mar 21, 2003 |
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09358856 |
Jul 22, 1999 |
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6537777 |
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60094258 |
Jul 27, 1998 |
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Current U.S.
Class: |
424/94.4 ;
435/325; 435/69.1; 514/44R |
Current CPC
Class: |
C12N 9/88 20130101; C12Y
205/01061 20130101 |
Class at
Publication: |
424/94.4 ;
514/44; 435/69.1; 435/325 |
International
Class: |
A61K 048/00; A61K
038/44; C12P 021/02; C12N 005/06 |
Claims
1. A method for treatment or prophylaxis of disease caused by
deficiency, in a subject, of one or more enzymes belonging to the
heme biosynthetic pathway, the method comprising administering, to
the subject, an effective amount of one or more catalysts which
is/are said enzyme(s), 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, wherein the catalyst is one or
more enzymes selected from the group consisting of
delta-aminolevulininic acid synthetase, delta-aminolevulinic acid
dehydratase (ALAD), porphobilinogen deaminase (PBGD),
uroporphyrinogen III cosythetase, uroporphyrinogen decarboxylase,
coproporphyrinogen oxidase, protoporphyrinogen oxidase, and
ferrochelatase, or an enzymatically equivalent part or analogue
thereof.
4. A method according to claim 1, wherein the disease is AIP and
the enzyme is PBGD or an enzymatically equivalent part or analogue
thereof.
5. A method according to claim 1, 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.
6. A method according to claim 1, 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.
7. A method according to claim 1, wherein the catalyst is
formulated in an isotonic solution.
8. A method according to claim 7, wherein the catalyst is
lyophilised.
9. A method according to claim 8, wherein the catalyst is sterile
filtered.
10. A method according to claim 1, wherein the catalyst is
formulated as lipid vesicles comprising phosphatidylcholine or
phosphatidylethanolamine or combinations thereof.
11. A method according to claim 1, wherein the catalyst is
incorporated into erythrocyte ghosts.
12. A method according to claim 1, wherein the catalyst is
formulated as a sustained release formulation involving
biodegradable microspheres.
13. A method according to claim 1, 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 13, 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.
15. A method according to claim 1, wherein the catalyst is
formulated in a physiological buffer containing an enhancer for
nasal administration.
16. A method according to claim 1, 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 claim 1, 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 17, wherein the catalyst is
complexed with a heavy metal.
20. A method according to claim 1, 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 claim 1, 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 claim 1, 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 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 claim 1, 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.
27. A method according to claim 1, wherein the catalyst has been
prepared by liquid-phase or solid-phase peptide synthesis.
28. A method according to claim 26 wherein the catalyst is free
from any other biological material of human origin.
29. A method according to claim 1, wherein the catalyst is
administered at least once a day.
30. A method according to claim 29 wherein the daily dosage is in
the range of 0.01-1.0 mg/kg body weight per day.
31. A method according to claim 29, wherein the daily dosage is
about 0.1 mg per kg body weight per day.
32. A method according to claims 1 wherein the catalyst is a
recombinant form of the enzyme.
33. A method according to claim 32 wherein the catalyst is
recombinant human PBGD based on any of Seq. ID NO 3 (clone PBGD
1.1) and Seq. ID NO 4 (non-erythro PBGD 1.1.1).
34. 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 whether the tissue in which PBGD should be
expressed is in cells of erythroid origin or in other cells, and
transfection of the patient with the relevant cDNA.
35. The method according to claim 34, 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).
36. The method according to claim 35 wherein the disease is Acute
Intermittent Porphyria, (AIP).
37. The method according to claim 34, wherein the human PBGD cDNA
sequence is selected from Seq. ID NO 3 (clone PBGD 1.1) and Seq. ID
NO 4 (non-erythro PBGD 1.1.1).
38. The method according to claim 34 wherein the transfection is by
use of a vector selected from the group consisting of adenovirus,
retrovirus and associated adenovirus vectors.
39. The method according to claim 34 wherein the PBGD transfection
of the patient (erythropoeitic and/or non-erythropoietic cells)
results in substantially normal PBGD activity measured as a
normalisation in urinary and/or serum levels of
delta-aminolevulinic acid (ALA) and porphobilonogen (PBG) compared
to the levels before treatment or to a reduction in the frequency
of attack of symptons.
40. 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.
41. The method according to claim 40-comprising a delivery system
for transfection which is by use of non-viral vectors formulated in
a vehicle preparation comprisng one or more components selected
from the group consisting of cationic phospholipids, phospholipids,
phospholipids mixed with neutral lipids, lictosylated PEI,
liposomes liposomes comprising mixtures of natural phopholipids and
neutral lipids.
42. A method according to claim 40 wherein the mutation is selected
from Table A.
43. A pharmaceutically acceptable composition comprising a
therapeutically or prophylactically effective amount of catalyst
which is an enzyme of the heme biosynthetic pathway or an
enzymatically equivalent part or analogue thereof.
44. A composition according to claim 43, wherein the catalyst is
recombinant human PBGD based on any of Seq. ID NO 3 (clone PBGD
1.1) and Seq. ID NO 4 (non-erythro PBGD 1.1.1).
Description
FIELD OF THE INVENTION
[0001] 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 alleviating
the symptoms of certain porphyrias, notably acute intermittent
porphyria including gene therapy, therapy wiht a combination of
encymatically active substances and therapy with recombinant
produced enzymes such as PBGD. In addition the invention relates to
an expression plasmid and a linear DNA fragment for use in the
production of rhPBGD.
BACKGROUND OF THE INVENTION
[0002] Heme Biosynthetic Pathway
[0003] 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) during oxidative phosphorylation in
mitochondria.
[0004] Heme is synthesised in eight consecutive enzymatic steps
starting with glycin and succinyl-CoA. Sassa S. 1996, Blood Review,
10, 53-58 shows a schematic drawing (FIG. 1 in the article) of the
heme biosynthetic pathway indicating that that the first enzymatic
step (delta-aminolevulinic-synthetase) and the last three steps
(coproporphyrinogen oxidase, protoporphyrinogen oxidase and
ferrochelatase) are located in the mitochondrion whereas, the
remaining are cytosolic enzymes.
[0005] Important regulation of the heme biosynthetic pathway is
delivered by the end product of the metabolic pathway, namely heme,
which exerts a negative inhibition on the first rate-limiting
enzymatic step (conducted by delta-aminolevulinic-synthetase) in
the heme biosynthetic pathway (Strand et al. 1970, Proc. Natl.
Acad. Sci. 67, 1315-1320).
[0006] Deficiencies in the heme biosynthetic enzymes have been
reported leading to a group of diseases collectively called
porphyrias.
[0007] A defect in the third enzymatic step leads to acute
intermittent porphyria, AIP.
[0008] Acute Intermittent Porphyria
[0009] 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. (Waldenstrom 1937, J. Acta.Med.
Scand. Suppl.82). In the following, this enzyme and the recombinant
human form will be termed "PBGD" and "rhPBGD", respectively.
[0010] Clinical Manifestation of AIP
[0011] 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).
[0012] 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 PBG and
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.
[0013] 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. 1996, American J. of Medical Genetics.
65, 269-273).
[0014] 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 behavioral changes, confusion,
seizures, respiratory paralysis, coma and hallucinations may be
present.
[0015] 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. 215, 271-274), has been
reported.
[0016] The AIP is a lifelong disease, which usually becomes
manifest in puberty.
[0017] Factors Precipitating Acute Attacks.
[0018] 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,
191-202).
[0019] 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).
[0020] 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.
[0021] Prevalence of AIP
[0022] 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 1500 in geographic isolates in northern Sweden (Wetterberg L.
1967, Svenska bokforlaget Nordstedt, Stockholm). Prevalence up to
200 per 10,000 inhabitants has been reported from Arjepong in
Northern Sweden (Andersson, Christer, Thesis, 1997, ISBN
91/7191/280/0, pp. 22-23).
[0023] Existing Treatment of AIP
[0024] 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.
[0025] Treatment between acute attacks involves sufficient caloric
intake and avoidance of drugs and immediate treatment of
infections.
[0026] Patients that experience acute attacks are treated with
intravenous carbohydrates usually dextrose (300 g/day) and
intravenous hematin (3-8 mg/(kg day)).
[0027] 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.
[0028] Medical Need in AIP
[0029] The lack of effective treatment for AIP is well recognized.
In a US mortality study in AIP patients requiring hospitalization
it was concluded that the mortality rate was 3.2-fold higher as
compared to a matched general population.
[0030] 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).
[0031] 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. 1989, Sem. Hematol.
26, 1-9).
[0032] 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 1989, Lancet 1,1295-1297).
[0033] In conclusion, there is a definite need for the provision of
novel therapeutic/prophylactic methods aimed at these diseases.
DISCLOSURE OF THE INVENTION
[0034] 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
.mu.g %) and PBG (334 .mu.g %) 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.
[0035] The present inventors present a new therapeutic rational in
the treatment of AIP, a rationale using PBGD, preferably
recombinant human PBGD (rhPBGD), in order to reduce circulating
high levels of PBG in serum by metabolizing (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.
[0036] 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.
[0037] Hence, PBGD administered by injections will carry out its
normal catalytic function by converting PBG to HMB in serum
(extracellularly, not inside the cells). The new therapeutic idea
is based on the assumption that ALA, PBG and HMB permeate cellular
membranes or is 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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 that mimic the specific enzymatic
activity of the relevant enzyme would also constitute "enzymatic
equivalent analogues".
[0043] 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.
[0044] The term "the heme biosynthetic pathway" refers to the
well-known enzymatic steps (cf. e.g. Sassa S. 1996, Blood Review,
10, 53-58) which leads from glycin and succinyl-CoA to heme, and
enzymes belonging to this synthetic pathway are
delta-aminolevulininic acid synthetase, delta-aminolevulinic acid
dehydratase, porphobilinogen deaminase, uroporphyrinogen III
cosythetase, uroporphyrinogen decarboxylase, coproporphyrinogen
oxidase, protoporphyrinogen oxidase and ferrochelatase. 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 the genes encoding all of the
above-mentioned enzymes have been sequenced, thus allowing
recombinant or synthetic production thereof.
[0045] 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).
[0046] 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).
[0047] 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.
[0048] 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.
[0049] 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 compartment. 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.
[0050] 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.
[0051] As mentioned above, it is preferred that the catalyst is
produced recombinantly, i.e. by a method comprising
[0052] a) introducing, into a suitable vector, a nucleic acid
fragment which includes a nucleic acid sequence encoding the
catalyst;
[0053] b) transforming a compatible host cell with the vector;
[0054] c) culturing the transformed host cell under conditions
facilitating expression of the nucleic acid sequence; and
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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 or combinations of enzymes and an
individualized treatment, where the dose is adjusted to normalize
patient serum and urine precusor levels.
[0060] 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.
[0061] 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 or
combinations of enzymes.
[0062] 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 or combination
of different catalysts for the preparation of pharmaceutical
compositions for treatment of the above-discussed diseases is also
part of the invention.
LEGENDS TO FIGURES
[0063] FIG. 1: Circular map of plasmid pPBGD1.1
[0064] FIG. 2: Flow chart for construction of plasmid pExp0
[0065] FIG. 3: Circular map of plasmid pExp0
[0066] FIG. 4: Flow chart for construction of plasmid pExp1
[0067] FIG. 5: Circular map of plasmid pExp1
[0068] FIG. 6: Flow chart for construction of pExp1-M2
[0069] FIG. 7: Circular map of plasmid pExp1-M2
[0070] FIG. 8: Flow chart for construction of rhPBGD expression
plasmid pExp1-M2-BB
[0071] FIG. 9: Circular map of rhPBGD expression plasmid
pExp1-M2-BB
[0072] FIG. 10: PCR strategy for construction of the EcoR I-Hind
III linear DNA-fragment
[0073] FIG. 11: Structure of the EcoR I-Hind III linear
DNA-fragment used for transformation
[0074] FIG. 12: Respiration and growth data from fermentation PD14
with strain PBGD-2
[0075] FIG. 13: rhPBGD expression in fermentation PD14 with strain
PBGD-2
[0076] FIG. 14: Chromatography on DEAE-Sepharose FF (DEAE1)
[0077] FIG. 15: Chromatography on DEAE-Sepharose FF (DEAE2)
[0078] FIG. 16: Chromatography on Butyl-Sepharose 4 FF
[0079] FIG. 17: Circular map of rhPBGD-His expression plasmid
pExp2
[0080] FIG. 18: PBGD reaction mechanism
[0081] FIG. 19: DEAE chromatography elution profile
[0082] FIG. 20: SDS-PAGE gel of DEAE eluates
[0083] FIG. 21: Cobalt chromatography elution profile
[0084] FIG. 22: SDS-PAGE gel results of cobalt eluates
SEQUENCE LIST
[0085] Seq. ID NO 1: Sequence of the expression plasmid
pExp1-M2-BB
[0086] Seq. ID NO 2: Sequence of the EcoR I-Hind III linear
fragment used for transformation in the hemC disruption
strategy
[0087] Seq. ID NO 3: Sequence of the erythropoietic form (PBGD
1.1)
[0088] Seq. ID NO 4: Sequence of the non-erythropoietic form (PBGD
1.1.1)
[0089] Seq. ID NO 5: Sequence of PDGB from Spleen (PBGD 1.3)
[0090] Seq. ID NO 6: Sequence of PDGB from bone marrow (PBGD
2.1)
[0091] Seq. ID NO 7: Sequence of PDGB from bone marrow (PBGD
2.2)
[0092] Seq. ID NO 8: Sequence of PDGB from lymph node (PBGD
3.1)
[0093] Seq. ID NO 9: Sequence of PDGB from lymph node (PBGD
3.3)
[0094] Seq. ID NO 10: Sequence PDGB from total brain (PBGD 5.3)
[0095] Seq. ID NO 11: Sequence of PDGB from total brain (PBGD
6.1)
DETAILED DISCLOSURE OF THE INVENTION
[0096] In a first embodiment the invention relates to a method for
treatment or prophylaxis of disease caused by deficiency, in a
subject, of one or more enzymes belonging to the heme biosynthetic
pathway, the method comprising administering, to the subject, an
effective amount of a catalyst which is said enzyme or combination
of enzymes 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
[0097] delta-aminolevulininic acid synthetase,
[0098] delta-aminolevulinic acid dehydratase (ALAD),
[0099] porphobilinogen deaminase (PBGD),
[0100] uroporphyrinogen III cosythetase,
[0101] uroporphyrinogen decarboxylase,
[0102] coproporphyrinogen oxidase,
[0103] protoporphyrinogen oxidase, and
[0104] ferrochelatase,
[0105] or an enzymatically equivalent part or analogue thereof.
[0106] The invention also relates to any combination of the enzymes
mentioned above because one enzymatic deficiency may cause such
alterations of the pathway that alternative enzymatic reactions are
needed wherein an otherwise normal production of an enzyme for such
alternative pathway is not sufficient. In alternative, the disease
relating to the heme biosynthetic pathway may also be due to a
deficiency of more than only one enzyme. Accordingly, en the
present context the term catalyst may also be interpreted as a
combination of catalyst and the term enzyme may also include a
mixture of different enzymes.
[0107] In a preferred embodiment, the disease is AIP and the enzyme
is PBGD or an enzymatically equivalent part or analogue thereof
optionally in combination with ALAD. 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.
[0108] 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.
[0109] The catalyst is preferable formulated in an isotonic
solution, such as 0.9% NaCl 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
lyophilized, 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.
[0110] Also a sustained release formulation may be performed
involving biodegradable microspheres, such as microspheres
comprising polylactic acid, polyglycolic acid or mixtures of
these.
[0111] 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.
[0112] It may also be very convenient to administer the catalyst in
a formulation of a physiological buffer containing an enhancer for
nasal administration.
[0113] Other formulations for the catalyst include an oral
formulation containing lipid vesicles, such as those comprising
phospatidylcholine, phosphatidylethanolamine, or sphingomyeline, or
dextrane microspheres.
[0114] The formulation is preferable one that 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.
[0115] The catalyst may also be complexed with a heavy metal.
[0116] 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 that can polymerise porphobilinogen
to hydroxymethylbilane.
[0117] 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.
[0118] In addition the catalyst may be tagged with specific
carbohydrates or other liver cell specific structures for specific
liver uptake.
[0119] In a furhter aspect the catalyst exerts substantially all
its enzymatic activity extracellularly in the bloodstream.
[0120] 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.
[0121] A further embodiment of the invention relates to a method
wherein the catalyst has been prepared by a method comprising
[0122] a) introducing, into a suitable vector, a nucleic acid
fragment which includes a nucleic acid sequence encoding the
catalyst;
[0123] b) transforming a compatible host cell with the vector;
[0124] c) culturing the transformed host cell under conditions
facilitating expression of the nucleic acid sequence; and
[0125] 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.
[0126] 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.
[0127] 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
treatment for evaluation of the specific dosage.
[0128] 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.
[0129] It is estimated that a dosage will often be about 0.1 mg per
kg body weight per day.
[0130] 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.
[0131] Naturally, the catalyst may be a recombinant form of the
enzyme. An example is a recombinant human PBGD based on any of Seq.
ID NO 3 and Seq. ID NO 4.
[0132] 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 deficiency is
selected from enzyme deficiencies 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).
[0133] In a preferred embodiment, the human PBGD cDNA sequence is
selected from Seq. ID NO 3 and Seq. ID NO 4.
[0134] The transfection may be by use of a vector 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.
[0135] 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 oligonucleotides and a specific knowledge of both
the mutation to be corrected and to the sequence on both sides on
the mutation.
[0136] 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.
[0137] The mutation may be selected from the mutations shown in
Table A.
[0138] 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.
[0139] Production of Recombinant Human PBGD (rhPBGD)
[0140] As mentioned above, it is preferred to administer
recombinant human 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.
[0141] The gene for the erythropoietic PBGD, which is located in
the human genome in the chromosomal region 11q24, is composed of 15
exons spanning 10 kb of DNA and is shown in Grandchamp B. et al.
1996, J. of Gastroenerology and Hepatology 11, 1046-1052.
[0142] 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 use of a
nested PCR (polymerase chain reaction) strategy.
[0143] The PBGD coding region will be inserted in a plasmid and
transformed into a suitable host cell (a bacterium such as E. coli
and B. subtilis, or a fungus such as Saccharomyces). The expression
of the PBGD gene will be regulated by a promoter which is
compatible with the selected host cell.
[0144] For bacterial production: An endogenous ATG sequence is
located at the NH.sub.2-terminal end of the PBGD 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.
[0145] The plasmid used for production of rhPBGD in E. coli was
constructed in the following way:
[0146] Construction of the Plasmid Harboring the Coding Region of
Human Wild Type PBGD (pBPGD1.1)
[0147] Introduction:
[0148] The erythropoietic expressed form of porphobilinogen
deaminase (PBGD) (Raich N. et al. 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. 1987, Eur J Biochem.
162(1):105-10). 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 H W. et al. 1993,
Genomics. 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 R. K. et al. 1985, Science
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.
[0149] Strategy:
[0150] A nested PCR strategy was devised to clone PBGD. The first
primer set, (see Table 1) Ico379 and Ico382, are 20mers that bind
to sequence outside of the coding region. Ico379 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. Ico382 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, Ico375 and Ico376, designed to distal ends of the
PBGD coding region. Ico375 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 EcoR I endonuclease
cleavage site for cloning of the PCR product and 4 bases of
sequence to ensure efficient restriction. Ico376 has 33 bases
homologous to the 3' end of the PBGD coding region with 3 bases
changed internally to introduce a Mun I/Mfe I 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 Hind III
endonuclease cleavage site for cloning the PCR product followed by
4 bases to ensure efficient restriction. The EcoR I and Hind III
endonuclease cleavage sites introduced onto the ends of the PBGD
PCR product ligate into the respective unique restriction sites in
the high copy number pBluescriptII 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, rhPBGD.
[0151] PCR:
[0152] 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 .mu.l reaction
volumes per manufacturers instructions except adipose which was
made from 5 .mu.g of Clontech total RNA from a single donor). List
of equipment and supplies used (see lists below). One .mu.l of each
cDNA (approximately 25 ng) was amplified with Advantage cDNA
polymerase mix (Clontech) with 0.2 mM dNTP (PE/ABI) and 0.3 .mu.M
each of Ico379 and Ico382 in 50 .mu.l reaction volumes. Two cycle
PCR was used, with an initial heat denaturation step at 94.degree.
C. for 1'40" then 28 cycles of 96.degree. C. for 16" and 68.degree.
C. for 2'. A final extension of 6' at 74.degree. C. ensured that
extension products were filled out. One fifth of the reaction was
run out on a 1.2% agarose gel with 2 .mu.l of 6.times. ficol
loading dye in 0.5.times. TBE buffer (Maniatis T., E. F. Fritsch,
J. Sambrook. Molecular Cloning (A laboratory Manual) Cold Spring
Harbor Laboratory. 1982). 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 dH.sub.2O. One
tenth of the recovered DNA was amplified with Advantage cDNA
polymerase mix (Clontech) with 0.2 mM dNTP (PE/ABI) and 0.3 .mu.M
each of the internal nested oligonucleotides (Ico375 and Ico376) at
0.3 .mu.M in 50 .mu.l reactions. Two cycle PCR was used again with
an initial heat denaturation step at 94.degree. C. for 1' 40" then
2 cycles of 96.degree. C. for 16" and 68.degree. C. for 2' then 13
cycles of 96.degree. C. for 16" and 72.degree. C. for 2' with a
final extension of 6' at 74.degree. C. Ten .mu.l of the 50 .mu.l
reactions were run on a 1.2% agarose gel with 2 .mu.l 6.times.
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 (10 mM
Tris-HCl pH8.0/1 mM EDTA). The DNA containing eluates were washed
with dH.sub.2O and concentrated with Microcon-100 spin-filters
(Amicon/Millipore) as described by the manufacturer's
instructions.
[0153] Cloning:
[0154] The purified PBGD DNA was digested for 6 hours with 40 Units
each of EcoR I and Hind III in EcoR I "U" buffer (New England
Biolabs (NEB)) in 50 .mu.l reactions at 37.degree. C. Enzymes were
heat killed for 20 minutes at 68.degree. C. and reactions were spun
in Microcon 100 spin-filters to remove small DNA end pieces, washed
with dH.sub.2O and concentrated. One fifth of the resulting DNA was
ligated with approximately 50 ng EcoR I and Hind III digested and
twice gel purified pBluescriptII SK-(Stratagene) and 200 units T4
DNA ligase (NEB cohesive end units) for 15 hours at 16.degree. C.
The ligase was heat killed at 75.degree. C. for 10 minutes. The
reactions were then buffer exchanged with dH.sub.2O and
concentrated in Microcon-100 spin filters and volumes taken up to 5
.mu.l with dH.sub.2O. One .mu.l each was electroporated into 25
.mu.l DH10B Electromax cells (Gibco/BRL) at 2.5 Kv/200 Ohms/25
.mu.F 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.degree. C. 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
.mu.g/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 specific primer (ICO381) was
used with a pBluescript specific primer (ICO385) to both confirm
identity and proper orientation in the vector. The 25 .mu.l
reactions were set up on ice to inactivate proteases with primer
concentrations of 0.4 .mu.M, 0.125U Taq polymerase (Fisher), and
0.2 mM dNTP(PE/ABI.) Two cycle PCR was used, with an initial heat
denaturation step at 94.degree. C. for 1' 40" a further denaturing
step at 96.degree. C. for 20 seconds, then 30 cycles of 96.degree.
C. for 16" and 68.degree. C. for 1' with a final extension of 4'at
74.degree. C. Five .mu.l of 6.times. loading dye was added and 12.5
.mu.l each were run out on a 1.2% agarose gel. Results are as
follows: 12/18 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 ml. liquid LB culture with 150
.mu.g/ml ampicillin over night at 37.degree. 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 260 nm was used to determine the plasmid yields which varied
from between 131 and 169 .mu.g of highly purified DNA.
[0155] Sequencing:
[0156] 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
(ICO383 and ICO384) and two PBGD specific internal primers (ICO380
and ICO381) were used for all 8 plasmids. In addition a fifth
vector primer (ICO385) 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 .mu.l ready mix in 20 .mu.l volumes with 30 cycles of 96.degree.
C. for 12" and 60.degree. C. for 4'. Extension products were
purified by isopropanol precipitation. To each reaction 20 .mu.l of
dH.sub.2O and 60 .mu.l isopropanol were added. These were mixed by
inversion and allowed to sit at room temperature for 15 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 1' 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.
[0157] Analysis:
[0158] 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 11 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 11 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.
[0159] Conclusion:
[0160] 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 deemed 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 (rhPBGD). In the following, the plasmid
encoding the human wild type PBGD in clone number 1.1 will be
termed "pPBGD1.1".
[0161] Equipment and supplies lists are shown in appendix 1 and 2,
respectively.
1TABLE 1 Oligonucleotide primers used for PCR amplification and
sequencing of PBGD: Ico375-pbgds (32 mer) coding region 5' end
w/EcoRI site sense 5'CGT GGA ATT CAT GAG AGT GAT TCG CGT GGG TA 3'
Ico376-pbgda (47 mer) coding region 3'end w/HindIII site antisense
5'GGA GAA GCT TAT TAA TGG GCA TCG TTC AAT TGC CGT GCA ACA TCC AG 3'
Ico379-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' Ico381-ainter (21 mer)
internal coding antisense 5'GAT GGC ACT GAA CTC CTG CTG 3'
Ico382-anonc (20 mer) non-coding antisense 5'CAG CAA CCC AGG CAT
CTG TG 3' Ico383-pSKT7 (22 mer) pBluescript T7 promoter 5'GTA ATA
CGA CTC ACT ATA GGG C 3' Ico384-pSKpjrev (22 mer) pBluescript
reverse1 5'CTA AAG GGA ACA AAA GCT GGA G 3' Ico385-pSKrev (21 mer)
pBluescript reverse2 5'CAG CTA TGA CCA TGA TTA CGC 3'
[0162]
2TABLE 2 Variation of PBGD clones from published erythroid
sequence: Differences from Erythroid mRNA non- total Genebank PBGD
clone silent silent diffs No. Reference/Source Erythroid 0 0 0
X04217 Raich. N. et. al. 1986, Nucleic Acids Res. 14 (15),
5955-5968 Constitutive 1 2 3 X04808 Grandchamp. B. et. al. 1987,
Eur. J. Biochem. 162 (1), 105-110 Genomic 1 2 3 M95623 Yoo, H. W.
et. al. 1993, Genomics 15 (1), 21-29 1.1 1 1 2 -- Spleen (Clontech
mRNA Lot No. 7120266) 1.3 2 1 3 -- Spleen (Clontech mRNA) 2.1 2 1 3
-- Bone Marrow (Clontech mRNA) 2.2 2 2 4 -- Bone Marrow (Clontech
mRNA) 3.1 2 4 6 -- Lymph Node (Clontech mRNA) 3.3 3 1 4 -- Lymph
Node (Clontech mRNA) 5.3 2 1 3 -- Total Brain (Clontech mRNA) 6.1 3
2 5 -- Adipose Tissue (Clontech mRNA)
[0163] Table 2:
[0164] Summary of the number of differences in amino acid sequence
of our sequenced PBGD clones and clones from Genebank entries 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 table 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 (rhPBGD) has only one of each type of difference with the
least number of total differences.
3TABLE 3 Summary of mutations found in PBGD clones: aa aa No. bp
No. mutation aa change cons. gen. 1.1 1.3 2.1 2.2 3.1 3.3 5.3 6.1
No./10 Asp 19 56 A.fwdarw.G Asp.fwdarw.Gly X 1 Phe 108 322 T
deletion frame shift X 1 Lys 140 419 A.fwdarw.G Lys.fwdarw.Arg X 1
Leu 160 478 C.fwdarw.A Leu.fwdarw.Met X 1 Ala 168 503 C.fwdarw.T
Ala.fwdarw.Val X 1 Leu 171 513 A.fwdarw.G silent X X X X X X X X X
9 Val 185 555 T.fwdarw.G silent X X X X X X X 7 Glu 193 577
G.fwdarw.A Glu.fwdarw.Lys X 1 Gly 243 729 C.fwdarw.T silent X 1 Ala
280 840 T.fwdarw.C silent X 1 Ala 286 856 G.fwdarw.A Ala.fwdarw.Thr
X 1 Lys 328 984 A.fwdarw.G silent X 1 Thr 332 995 C.fwdarw.A
Thr.fwdarw.Asn X X X X X X X X X X 10 Gln 339 1017 G.fwdarw.A
silent X X X X X X X X 8 Gln 339 1018 C.fwdarw.T silent X X X X X X
X X 8 Leu 340 1020 T.fwdarw.G silent X X X X X X X 7 Leu 340 1020 T
deletion frame shift X 1 Table 3: Summary of the genetic
differences of our sequenced PBGD clones and Genbank entries 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. In the final column the total number of clones
with the different mutations are shown. The three mutations at
position 1017, 1018 and 1020 are introduced with IC0376 during PCR
amplification to create a Mun 1 endonuclease cleavage site. # Note
that clone number 1.1 which will be used for represented by a
number of other clones.
[0165] Expression Plasmids
[0166] Construction of the Basic Expression Plasmid pExp0
[0167] The basic expression plasmid pExp0 was constructed by
excising the PBGD coding sequence (cDNA) from plasmid pPBGD1.1 (see
FIG. 1) with EcoR I and Hind III and inserting it into the vector
pKK223-3 (Pharmacia, Catalogue #27-4935) cut with the same enzymes,
thus operatively linking it to the IPTG-inducible tac promoter
(Amann E. et al. 1983, Gene 25(2-3):167-178). FIG. 1 shows the
construction details. Plasmid pExp0 was constructed for a
preliminary assessment of the expression levels and does not
directly lead to the construction of the final expression
plasmid.
[0168] Construction of the Final Expression Plasmid
[0169] The final expression plasmid pExp1-M2-BB (FIG. 9) was
constructed in a multi-step process. The individual steps used and
all the intermediate plasmids are outlined below.
[0170] Construction of Plasmid pExp1
[0171] Plasmid pExp1 was first constructed with modifications to
the 5' untranslated region and the initial part of the coding
sequence both aimed at improving translation efficiencies (Gold L.
and Stormo G. D. 1990, Methods Enzymol 185:89-93). 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 boldface.
4 AATTCTAACA TAAGTTAAGG AGGAAAAAAA A ATG AGA GTT ATT CGT GTC GGT AC
Met-Arg-Val-Ile-Arg-Val-Gly
[0172] A naturally occurring Kpn I site six amino acid residues
into the coding sequence of the human cDNA for PBGD (pPBGD1.1) was
exploited for this purpose. Oligonucleotides ICO386 and ICO387 were
designed to provide upon annealing to each other a 5' EcoR I
adhesive end and a 3' Kpn I sticky end and the elements described
above including the codons for the first six amino acid residues as
shown. Oligonucleotides ICO386 and ICO387 were annealed and ligated
with the Kpn I-Hind III, PBGD fragment from pPBGD1.1 into EcoR
I-Hind III linearised pBluescript II SK-(Stratagene,
Catalogue#212206) to yield plasmid pPBGD1.1Tra. In the second step,
the EcoR I-Hind III fragment from pPBGD1.1Tra was ligated into
pKK223-3 cut with the same enzymes resulting in plasmid pExp1
(FIGS. 4 and 5).
[0173] Construction of Plasmid pExp1-M2
[0174] The tetracycline resistance gene was next restored using the
following strategy. Plasmid pExp1 was cut with Sal I and BamH I and
the 5349 base-pair fragment containing part of the tetracycline
coding sequence and the bulk of the plasmid was isolated. Into this
was ligated the Sal I-Hind III fragment from pBR322 (New England
BioLabs, Catalogue #303-3S) containing rest of the coding sequence
and an adapter formed by annealing oligonucleotides ICO424 and
ICO425 to each other. The adapter contains part of the tetracycline
promoter and provides Hind III and BamH I overhangs for ligation
but destroys the Hind III and BamH I restriction sites. The
resulting plasmid was called pExp1-M2 (FIGS. 6 and 7).
[0175] Construction of Plasmid pExp1-M2-BB
[0176] In the final step the rop gene contained between BsaA I and
BsaBI was deleted to increase copy number (Makrides S. C. 1996,
Microbiol.Rev. 60(3):512-538). For this the plasmid pExp1-M2 was
cycled through the dam minus strain, JM110 (F'[traD36 proA.sup.+
proB.sup.+ lacI.sup.q A (lacZ)M15] dam dcm supE44 hsdR17 thi leu
thr rpsL lacY galK galT ara tonA tsx .DELTA. (lac-proAB)
lambda.sup.-) as restriction with BsaB I is blocked by overlapping
dam methylation. It was then cut with BsaA I and BsaB I to excise
the rop gene and the 5446 base-pairs long linear fragment was
circularised by blunt-end ligation to yield the production plasmid
pExp1-M2-BB (FIGS. 8 and 9).
[0177] Construction of the hemC-Deletion Host and the Final
Expression Strain
[0178] The parent strain JM105 (F'[traD36 proA+proB+lacI.sup.q
.DELTA.(lacZ)M15] .DELTA.(pro-lac) hsdR4 sbcB15 rpsL thi endA1
lambda.sup.-), a derivative of E. coli K12 was obtained from
Parmacia, Catalogue #27-1550-01. The hemC gene coding for the
endogenous E. coli Porphobilinogen Deaminase was partially deleted.
This was necessary to ensure that the purified product (rhPBGD) was
free from contaminating E. coli PBGD as the E. coli and human
enzymes are very similar in properties (Jordan P. M. 1994, Wiley,
Chichester (Ciba Found Symp 180), p70-96) and may co-purify. The
hemC-deletion host was derived from JM105 according to Scheme A.
First a hemin-permeable variant was obtained by a three-step
process. This was essential as a hemC-deletion mutant would require
hemin for good growth and E. coli K12 strains are not freely
permeable to hemin. 1
[0179] All mutant isolation was spontaneous. Approximately
5.times.10.sup.-8-5.times.10.sup.-9 cells were plated on selective
media. The media compositions are included as Appendix 1.
[0180] * For details on the linear DNA-fragment see FIG. 11.
[0181] In the first step, a heme-minus mutant was isolated carrying
a defect in any of the biosynthetic steps leading to the formation
of heme. Heme.sup.- strains fall into the general class of
respiration deficient mutants that are defective in active
transport and consequently resistant to low levels of antibiotics
of the aminoglycoside family such as gentamicin (Lewis L. A. et al.
1991, Microbiol.Immunol. 35(4):289-301). Several spontaneous
mutants were isolated as a dwarf colonies on LB+glucose +G-418
(gentamicin)-containing plates (Lewis L. A. et al. 1991,
Microbiol.Immunol. 35(4):289-301). These were screened for their
ability to respond weakly to hemin, indicating that they were
heme.sup.- (as opposed to other respiration deficient mutants which
would not respond to hemin at all). One such heme-strain
(JM105-2-4, see Scheme A) which could also revert back
spontaneously to robust growth on LB (as this is essential for the
third step, see below) was selected. This strain was next plated on
LB+hemin to obtain a better grower in the presence of hemin and was
called JM105-H. It showed improved growth only in the presence of
hemin, which meant that it still was heme.sup.- but had become
hemin-permeable. To restore the functionality of the heme
biosynthetic pathway in JM105-H, spontaneous revertants were
isolated on LB plates and only those retained which resembled the
starting strain JM105 in growth, both untransformed and after
transformation with the expression plasmid. One such strain used in
this study was called JM105-H-R6 and should have retained the
heme-permeable trait of its parent strain.
[0182] Strain JM105-H-R6 was transformed with the EcoR I-Hind III
fragment (see Scheme A), to obtain the hemC-deletion host called
JM105-H-R6-C by homologous gene replacement. This strain has the
genotye, F'[traD36 proA+proB+lacI.sup.q .DELTA.(lacZ)M15]
.DELTA.(pro-lac) hsdR4 sbcB15 rpsL thi endA1 lambda.sup.- hemC:CAT
hemin-permeable. It was transformed with the expression plasmid
pExp1-M2-BB to yield the final production strain PBGD-2 (PBGD-2 was
deposited under the Budapest Treaty on Jul. 9, 1999 with DSMZ
(Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH,
Mascheroder Weg 1b, D-38124 Braunschweig, Germany) under the
accession No. DSM 12915).
[0183] In order to obtain the EcoR I-Hind III fragment, a multiple
PCR strategy was used. Olignucleotide pairs ICO437, 1C0438 and
ICO505, ICO440 were used to amplify separately, portions of E. coli
JM105 genomic DNA segments flanking the hemC gene (see FIG. 10).
These amplified gene products were digested with pairs of enzymes
EcoR I, Xho I and Xho I, Hind III respectively, and in essence,
assembled together between the EcoR I and Hind III sites of pUC19
to give plasmid phemCd. Next the fragment containing the
chloramphenicol-resistance gene was PCR amplified from plasmid pBC
SK+ (Stratagene, Catalogue #212215) using oligonucleotides ICO510
and ICO511. This product was cut with Xho I and inserted into
plasmid phemCd at the Xho I site. In essence, the plasmid having
the Cam gene in the orientation shown was called phemCdCm and
formed the source of the EcoR I-Hind III linear DNA-fragment
depicted in FIG. 11.
[0184] In FIG. 11 the structure of the linear DNA-fragment used for
the transformation is shown. The genetic organization of the E.
coli polypeptides depicted by gray arrows (cyaA, hemC-5', hemC-3',
hemX and hemD) is derived from the GenBank report Accession number
AE000456. The black arrow represents the 767 base-pairs long PCR
fragment carrying the cholamphenicol-resistance gene (Cam),
encoding chloamphenicol acetyltransferase (CAT), replacing 806
base-pairs of the hemC coding sequence. HemC-5' and hemC-3'
correspond to 149 and 16 base-pairs respectively, of the coding
sequence of the disrupted hemC gene. EcoR I, Xho I and Hind III are
engineered restriction sites. The sequence of this 3225 base-pairs
long fragment is shown in Seq. ID NO 2. The two Xho I sites are at
positions 1072 and 1839 in the sequence, respectively.
5TABLE 4 Oligonucleotide primers used in the construction of the
production strain PBGD-2 ICO386 (54 mer) Construction of plasmid
pExp1 5' AAT TCT AAC ATA AGT TAA GGA GGA AAA AAA AAT GAG AGT TAT
TCG TGT CGG TAC 3' ICO387 (46 mer) Construction of plasmid pExp1 5'
CGA CAC GAA TAA CTC TCA TTT TTT TTT CCT CCT TAA CTT ATG TTA G 3'
ICO424 (32 mer) Construction of plasmid pExp1-M2 5' GAT CAC TCA TGT
TTG ACA GCT TAT CAT CGA TT 3' ICO425 (31 mer) Construction of
plasmid pExp1-M2 5' AGC TAA TCG ATG ATA AGC GTC AAA CAT GAG T 3'
ICO437 (32 mer) Amplification of product P1 5' AGT CAG AAT TCA GAC
GCA CGG CGG TAC GAT AA 3' ICO438 (32 mer) Amplification of product
P1 5' ATT CAC TCG AGG TCA CCA TCG GTA CCA GTT CA 3' ICO440 (32 mer)
Amplification of product P2 5' AGA TCA AGC TTC GGC CAG ACG CAG GTT
ATC TA 3' ICO505 (34 mer) Amplification of product P2 5' ATA CAC
TCG AGA CCG GCA TGA GTA TCC TTG TCA C 3' ICO510 (30 mer)
Amplification of Cam gene 5' ACT GAC CTC GAG CGG CAC GTA AGA GGT
TCC 3' ICO511 (29 mer) Amplification of Cam gene 5' ACT GAA CTC GAG
AAT TAC GCC CCG CCC TG 3'
[0185] Accordingly, the cDNA used for expressing rhPBGD was derived
from plasmid pPBGD1.1.
[0186] The starting host strain was derived from JM105 and is
called JM105-H-R6-C. The genotype and the details on its
construction are described above. A large part of the coding region
of the hemC gene was replaced by the Cam gene, encoding
chloramphenicol acetyltransferase. This gene replacement was
confirmed by PCR amplification of the segment of the E. coli genome
followed by restriction analysis of the amplified product. As a
result of the gene replacement, the strain is resistant to
chloramphenicol and grows extremely poorly on LB medium. Growth
improves when LB medium is supplemented with hemin.
[0187] Expression Construct
[0188] The expression plasmid in the final production strain is
pExp1-M2-BB. Its construction is described above. A detailed map of
the plasmid showing the open reading frames and functionally
relevant regions is shown in FIG. 9. The complete DNA sequence is
included in Seq. ID NO 1. All synthetic adapters and linkers used
during the construction have been sequenced along with all
junctions created during ligation which directly impinge upon the
expression of the cloned gene.
[0189] Production Strain
[0190] The final production strain is called PBGD-2. It was
obtained by introducing the expression plasmid pExp1-M2-BB into the
host strain JM105-H-R6-C, essentially, by rendering the cells
competent with 100 mM CaCl.sub.2 (Morrison D. A. 1979,
Methods.Enzymol. 68:326-331) and selecting for transformants on
LB+ampicillin media at 30.degree. C. The plasmid is a derivative of
pBR322 without the rop gene and should be present
extrachromosomally in moderate copy number at 30.degree. C. with a
slightly higher copy number at elevated temperatures 37.degree. C.
and greater (Makrides S. C. 1996, Microbiol.Rev. 60(3):512-538). It
has both the ampicillin and tetracycline resistance genes as
selectable markers. It also expresses rhPBGD which can complement
the hemC defect of the host strain. As a result, the production
strain should grow normally on LB/M9 media, be resistant to the
antibiotics ampicillin and tetracycline and also be resistant to
the antibiotic chloramphenicol (because of the presence of the Cam
gene in the genome). It was confirmed to have all these
characteristics.
[0191] Expression
[0192] The expression of rhPBGD is driven by the tac promoter which
is regulated by the copy of the lacI.sup.q gene present in the
host. Due to the modifications made to the system as described in
the study plan, the uninduced level of expression is 1.8 units/mg
(see Appendix 3 for assay details), which amounts to approximately
10% of the total soluble protein. The culture is grown throughout
at 30.degree. C. and no induction step is used to increase
expression.
[0193] Evaluation and Conclusions
[0194] The expression system developed for the production of rhPBGD
in E. coli is a stable system, producing good amounts of rhPBGD in
a constitutive manner when the cells are grown at 30.degree. C. The
host strain employed is partially deleted for the gene producing
the endogenous E. coli porhobilinogen deaminase. After
transformation with the expression plasmid, the resulting
production strain PBGD-2 grows as well as the strain PBGD-1 (which
is JM105 carrying the same expression plasmid) and makes the same
amount of rhPBGD.
[0195] Alternative Expression Construct:
[0196] Expression Plasmid pExp1-M2-Puc-BB and Expression of rhPBGD
in E. coli
[0197] The 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 I-AflIII 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 and differs from pExp1-M2-BB only in that C in position
2769 is T in pExp1-M2-Puc-BB.
[0198] Expression of rhPBGD in E. coli
[0199] The E. coli K12 host strain JM105 genotype endA thi rpsL
sbcB15 hsdR4 .DELTA.(lac-proAB) [F'traD36 proAB lacI.sup.q
.DELTA.(lacZ)M15] containing the expression plasmid pExp1-M2-Puc-BB
was grown in LB broth containing 100 .mu.g/ml ampicillin at to
mid-log phase at 30.degree. 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.degree. C.,
37.degree. C. and 42.degree. C. respectively. Cells were spun down
from 1 ml samples and frozen at -20.degree. C. The thawed cell
pellets were resuspended in 200-300 .mu.l 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.degree. C. to 40.degree. C.
6 Temperature PBGD Units/mg 30.degree. C. 363 37.degree. C. 573
42.degree. C. 1080
[0200] Other Production Systems For rhPBGD
[0201] 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 trp1-, ura 3-yeast strains.
[0202] Alternatively, the PBGD gene can be inserted in bovine
papilloma virus vectors BPV for high expression in a murine cell
line C-127 (Stephens P. E. et. al. Biochem J. 248, 1-11, 1987) or
vectors compatible with expression in CHO cells or COS cells.
[0203] An expression of PBGD can be made intracellularly.
[0204] A secretory signal in Saccharomyces, for example
alpha-mating factor presequence, can be added in front of the
rhPBGD structural gene for efficient secretion in yeast.
[0205] Similarly, a sequence encoding a mammalian signal peptide
can be added for secretion of rhPBGD into the culture medium upon
expression in for example CHO cells or COS cells.
[0206] 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.
[0207] 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.
[0208] 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.
[0209] 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
rhPBGD.
[0210] 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) may be transfected into a mammalian cell
line for example CHO cells, for production of rhPBGD.
[0211] The E. coli cell containing plasmid (pExp1 or pExp1-M2
Puc-BB), may be fermented to stationary phase between 24-48 hours,
in a medium containing casein hydrolysate, or yeast extract,
glucose, vitamins and salts. pH oxygen may be monitored by
electrodes during fermentation. Temperature will be kept at 37+/-2
C. during the fermentation.
[0212] The yeast cell containing the plasmid (Y-G&F-PBGD), may
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.
[0213] The mammalian cell line containing the plasmid
(M-G&F-PBGD) may 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.
[0214] Fermentation and Purification
[0215] rhPBGD may be 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-100 or Brij. rhPBGD will be recovered
from fermentation medium after production in yeast or from a total
cellular extract using detergents such as Triton X-100, Tween 80 or
Brij. rhPBGD will be recovered from mammalian culture medium or
from a total cellular extract by ion-exchange chromatography or
affinity chromatography.
[0216] rhPBGD may 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 NaCl and Sodium phosphate buffer pH 7-8 or the
corresponding potassium salts.
[0217] Alternatively, rhPBGD may be recovered from extracts by
binding to an affinity chromatography column for example an
anti-PBGD affinity column. rhPBGD will be eluted by lowering the pH
to 4-2, or a thiol specific affinity column. rhPBGD 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 rhPBGD.
[0218] The ion-exchange or affinity purified rhPBGD will be further
purified by hydrophobic interaction chromatography on for example,
TSK Phenyl 5 PW column or Octyl-Sepharose or Phenyl-Sepharose
columns.
[0219] Binding of rhPBGD may be done at high ionic strength for
example in 10-50 mM Tris-HCl pH 7-8, 1M NaCl or 10-15 mM Sodium
phosphate pH 7-8, 0.5 M MgSO.sub.4 and eluted by lowering the ionic
strength for example with 10-50 mM Tris-HCl pH 7-8 or 10-50 mM
Sodium phosphate pH 7-8.
[0220] Three hydrophobic interaction steps will be applied
consecutively.
[0221] rhPBGD is further purified with preparative RP-HPLC for
example C12 or C18 matrixes. The rhPBGD is be eluted from the
column by a gradient of 10-50 mM Sodium phosphate and 1-10%
acetonitrile buffer.
[0222] Formulation of rhPBGD is done by passing the enzyme over a
G-100 Sephadex column and eluting it in an isotonic solution for
example 0.9% NaCl and 10-50 mM Sodium phosphate pH7.0+/-0.5 or
Sodium phosphate, glycin, mannitol or the corresponding potassium
salts.
[0223] For the preparation of a medicament, the formulation
solution of rhPBGD may be sterile filtered and filled aseptically
in glass vials and lyophilised.
[0224] Alternatively, the sterile filtered rhPBGD solution is
formulated in for example, lipid vesicles constituting
phosphatidylcholine or phosphatidylethanolamine or combinations of
these or incorporated into erythrocyte ghosts.
[0225] Reconstitution of lyophilised rhPBGD may be done in water
for injection.
[0226] Alternatively, rhPBGD is formulated in a sustained release
formulation involving a biodegradable microspheres, for example in
polylactic acid, polyglycolic acid or mixtures of these.
[0227] Alternatively, rhPBGD is lyophilized in a two-compartment
cartridge, where rhPBGD will be in the front compartment and water
for reconstitution in the rear compartment. This two compartment
cartridge may be combined with an injection device to administer
either rhPBGD by a needle or needle less (by high pressure)
device.
[0228] Alternatively, rhPBGD may be formulated in a physiological
buffer containing an enhancer for nasal administration.
[0229] Alternatively, rhPBGD is formulated in an oral formulation
containing for example, lipid vesicles (phospatidylcholine,
phosphatidylethanolamine, sphingomyeline) or dextrane
microspheres.
[0230] Although recombinant production of PBGD is preferred for the
treatment of AIP, it can alternatively be produced from human red
blood cells.
[0231] A general production and manufacturing of recombinant PBGD
may be done by the following steps.
[0232] Recombinant PBGD Production Process; an Outline
[0233] A: Fermentation
[0234] 1. Master cell bank
[0235] 2. Working cell bank
[0236] 3. Production of seed culture
[0237] 4. Fermentation in large fermenter (250 L>)
[0238] B. Purification
[0239] 1. Cell concentration by filtration/centrifugation
[0240] 2. Cell disruption
[0241] 3. Ultrafiltration
[0242] 4. Chromatography ion exchange, DEAE-Sepharose,
MonoQ-Sepharose
[0243] 5. Hydrophobic interaction chromatography
(Octyl/phenyl-Sepharose, TSK Phenyl, 5PW, Phenyl -Sepharose
[0244] 6. Chromatography ion exchange (MonoQ)
[0245] 7. Formulation by Gel filtration Sephadex G-100
[0246] C. Manufacturing
[0247] 1. Sterile filtration
[0248] 2. Aseptic filling
[0249] 3. Lyophilization
[0250] Treatment of Other Porphyrias
[0251] 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.
[0252] 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.
[0253] Hepatoerythropoietic Porphyrias e.g. Hepatoerythropoietic
Porphyrias(HEP) can be treated with rUroporphyrinogen
decarboxylase.
[0254] 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.
[0255] 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.
[0256] Gene Therapy as and Alternative Treatment for Patients wiht
Acute Intermittent Porphyria (AIP)
[0257] The human enzyme Porphobilinogen deaminase PBGD is coded for
by a single gene located on chromosome 11q24.
[0258] Mutations in this gene causes the disease Acute Intermittent
Porphyria (AIP). The disease has been shown to be inhereted in an
autosomal dominat way.
[0259] 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.
7TABLE A Reported mutations in the PBGD gene Position Mutation
Consequences Reference Exon 1 3 ATG.fwdarw.ATA Translation 18
impairment 33 GCG.fwdarw.GCT DS 17 Intron 1 33 + 1 gtg.fwdarw.atg
DS 16 Exon 3 76 CGC.fwdarw.TGC R26C 25 77 CGC.fwdarw.CAC R26H 26
Exon 4 91 GCT.fwdarw.CACT A31T 24 97 Del A Frameshift 25 100
CAG.fwdarw.AAG Q34K 27 100 CAG.fwdarw.TAG Q34X 25 125
TTG.fwdarw.TAG L42X 19 Exon 5 163 GCT.fwdarw.TCT A55S 24 174 Del C
Frameshift 24 182 Ins G Frameshift 24 Intron 5 210 + 1
gta.fwdarw.ata DS (Del exon 5) 24 Exon 6 218-219 Del AG Frameshift
24 Exon 7 277 GTT.fwdarw.TTT V93F 24 293 AAG.fwdarw.AGG K98R 25 331
GGA.fwdarw.AGA G111R 28 Intron 7 345 - 1 cag.fwdarw.caa AS (Del
exon 8) 29 Exon 8 346 CGG.fwdarw.TGG R116W 20 347 CGG.fwdarw.CAG
R116Q 30 Exon 9 445 CGA.fwdarw.TGA R149X 25 446 CGA.fwdarw.CAA
R149Q 31 446 CGA.fwdarw.CTA R149L 24 463 CAG.fwdarw.TAG Q155X 32
470 Ins A Frameshift 29 Intron 9 499 - 1 cag.fwdarw.caa AS (Del
exon 10) 21 Exon 10 499 CGG.fwdarw.TGG R167W 33 500 CGG.fwdarw.CAG
R167Q 27, 34 518 CGG.fwdarw.CAG R173Q 34 530 CTG.fwdarw.CGG L177R
27 593 TGG.fwdarw.TAG W198X 19 604 Del G Frameshift 35 610
CAG.fwdarw.TAG Q204X 30 612 CAG.fwdarw.CAT DS (Del 9 bp 31 exon 10)
Exon 11 625 GAG.fwdarw.AAG E209K 28 Intron 11 652 - 3
cag.fwdarw.gag AS (Del exon 12) 33 Exon 12 667 GAA.fwdarw.AAA E223K
24 673 CGA.fwdarw.GGA R225G 25 673 CGA.fwdarw.TGA R225X 25 713
CTG.fwdarw.CGG L238R 25 715-716 Del CA Frameshift 19 730-731 Del CT
Frameshift 36 734 CTT.fwdarw.CGT L245R 31 739 TGC.fwdarw.CGC C247R
36 740 TGC.fwdarw.TTC C247F 18 742 Ins 8 bp Frameshift 24 748
GAA.fwdarw.AAA E250K 24 754 GCC.fwdarw.ACC A252T 36 755
GCC.fwdarw.GTC A252V 36 766 CAC.fwdarw.AAC H256N 27 771
CTG.fwdarw.CTA DS (Del exon 12) 39 771 CTG.fwdarw.CTC DS (Del exon
12) 37 Intron 12 771 + 1 gta.fwdarw.ata DS (Del exon 12) 19 Exon 13
806 ACA.fwdarw.ATA T269I 30 820 GGG.fwdarw.AGG G274R 30 Exon 14 838
GGA.fwdarw.AGA G280R 25 848 TGG.fwdarw.TAG W283X 30 886
CAG.fwdarw.TAG Q296X 25 900 Del T Frameshift 31 Intron 14 912 + 1
gta.fwdarw.ata DS (Del exon 14) 28 Exon 15 1062 Ins C Frameshift 38
1073 Del A Frameshift 25
[0260] In one aspect, the present invention relates to a
therapeutic method for AIP patients based on gene therapy.
[0261] The gene therapy treatment may involve the following
steps.
[0262] 1. Identification mutations in the PBGD gene causing AIP in
humans
[0263] 2. Selection of human PBGD cDNA sequence for gene
therapy
[0264] 3. Construction of PBGD gene therapy vectors.
[0265] 4. Production of PBGD gene transfer vector
[0266] 5. Delivery system of PBGD gene transfer vector
[0267] 1. Identification of Mutations in the PBGD Gene Causing AIP
in Humans
[0268] 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 J S. et al.
Proc. NatI. 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.
[0269] 2. Selection of Human PBGD cDNA Sequence for Gene
Therapy
[0270] 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.
[0271] Non-Erythropoietic PBGD Form (nPBGD):
8
Met-Ser-Gly-Asn-Gly-Asn-Ala-Ala-Ala-Thr-Ala-Glu-Glu-Asn-Ser-Pro-L-
ys-Met-Arg-Val . . . ATG-TCT-GGT-AAC-GGC-ATT-GCG-GCT-GCA-ACG-GCG-G-
AA-GAA-AAC-AGC-CCA-AAG-ATG-AGA-GTG . . .
[0272] Erythropoietic PBGD Form (ePBGD):
9 Met-Arg-Val- ATG-AGA-GTG . . .
[0273] 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.
[0274] 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.
[0275] 3. Construction of PBGD Gene Therapy Vectors
[0276] Adenoviral Vector System
[0277] 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 E1B 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.
[0278] The human PBGD cDNA will be inserted in an adenovirus vector
system.
[0279] The PBGD transgenes will be driven by the endogenous PBGD
promoter or a cytolomega virus promoter (CMV).
[0280] Retroviral Vectors
[0281] Retroviral vectors are well suited for gene delivery for
several reasons:
[0282] 1. simplicity
[0283] 2. capacity to integrate up to 8 kbp DNA inserts
[0284] 3. their safety, non pathogenic to humans
[0285] 4. easy to improve and manipulate
[0286] 5. defined integration sites of genes
[0287] 6. long term regulated expression
[0288] One major disadvantage with the retroviral vectors though,
is that they can only transduce dividing cells.
[0289] 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).
[0290] 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.
[0291] 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.
[0292] LePSN: 2
[0293] 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. 3
[0294] The LePSN and LnPSN vectors can be converted to the
corresponding virus by transfer into an appropriate host cell line
e.g. .PSI. 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 .PSI. 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 pMCl-Neo plasmid (Pharmacia, Sweden) into the packaging cell
line .PSI. CRIP. Clones that shows integration of provirus and high
expression levels of message will be selected.
[0295] 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.
[0296] 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.
[0297] Associated Adenovirus System (AAV)
[0298] AAV is a non-pathogenic human virus (Parvovirus) carried by
more than 80% of all people.
[0299] 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.
[0300] Non-erythropoietic
[0301] 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.
[0302] 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.
[0303] Erythropoietic
[0304] 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.
[0305] 4. Production of PBGD Gene Transfer Vector
[0306] 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).
[0307] 5. Delivery Systems of PBGD Gene Transfer Vectors.
[0308] Delivery of viral vectors are based on injection into the
patient of a virus particle that will transduce human cells in
vivo.
[0309] Correction of Point Mutations Causing AIP by Chimeraplasty
Gene Repair
[0310] 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.
[0311] The Chimeric oligonucleotides has the following general
properties:
[0312] a. 68 mer (65-70 is acceptable size)
[0313] b. 25 base DNA stretches at the 5'-end homologous to the
normal sequence of the gene
[0314] 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
[0315] 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.
[0316] e. the second strand is chimeric in that it contains 10
homologous bases of 2'O 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 2'O 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 A
[0317] Correction of the PBGD Mutation at Position 593 TGG>TAG
Resulting in W198X
[0318] Normal Chromosomal Sequence:
10 5'-AG CGC ATG GGC TGG CAC AAC CGG GT-3' Gln Arg Met Gly Trp His
Asn Arg Val
[0319] AIP Chromosomal Sequence:
11 5'-AG CGC ATG GGC TAG CAC AAC CGG GT-3' Stop
[0320] The sequence of the chimeric oligonucleotide ( Heme593W/X)
is: 4
[0321] The same principle of chimeric oligonucleotide can be
constructed to correct any of the mutations causing AIP depicted in
Table A.
[0322] Chimeric oligonucleotides can be used to correct any other
point mutation causing any of the 8 known Porphyrias in a similarly
as described above.
[0323] Delivery of PBGD Gene Transfer of Non Viral Vectors to
Humans
[0324] The chimeric oligonucleotide can be formulated in a
vechicles preparation containing anionic or cationic phospholipids
or phospholipids mixed with neutral lipids or lictosylated PEI.
[0325] Alternatively, the non-viral vectors can be formulated in
liposomes containing mixtures of natural phospholipids and neutral
lipids.
[0326] Specific protein sequences 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.
[0327] The formulated chimeric oligonucleotide (HemeBiotech 595
W/X) will be administered by sc. or IV. injections to AIP
patients.
[0328] The efficacy of the treatment can be evaluated as above.
[0329] Gene Therapy as an Alternative Treatment of Other Porphyric
Diseases
[0330] 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:
[0331] 1. ALA deficiency porphyria (ADP)
[0332] 2. Porphyria cutanea tarda (PCT)
[0333] 3. Hereditary coproporphyria (HCP)
[0334] 4. Harderoporphyria (HDP)
[0335] 5. Variegata porphyria (VP)
[0336] 6. Congenital erythropoietic porphyria (CEP)
[0337] 7. Erythropoietic protoporphyria (EPP)
[0338] 8. Hepatoerythropoietic porphyria (HEP)
[0339] In the following Examples, preferred embodiments of the
invention is disclosed relating to rhPBGD
EXAMPLE 1
[0340] Fermentation of Recombinant Human Porphobilinogen Deaminase
(rhPBGD)
[0341] Strain PBGD-1 is an E. coli K12 host strain JM105 genotype
endA thi rpsL sbcB15 hsdR4 .DELTA.(lac-proAB) [F'traD36 proAB
lacI.sup.q .DELTA.(lacZ)M15] containing the expression plasmid
pExp1-M2-BB. Strain PBGD-2 has the same expression plasmid
pExp1-M2-BB but the host cell is deleted for the hemc gene to
facilitate rhPBGD purification. Since the strain PBGD-2 was not
ready at the start of the study, the decision was made to start the
study with strain PBGD-1. Both the strains are resistant against
both tetracycline and ampicillin, but due to regulatory advantages
it was decided to use oxytetracycline as selection pressure. To
focus the first part of the study on the expression level of
rhPBGD, and not the strain stability, it was decided to start the
development with selection pressure in the fermenter. When the
expression level was satisfactory strain stability without
selection pressure in the fermenter should be investigated.
Preliminary tests performed showed that the expression level of
rhPBGD was 1,5 times higher at 37.degree. C. compared to the
expression at 30.degree. C. At 42.degree. C. it was as much as 3
times higher. Based on this knowledge one would suggest a
temperature induction to either 37.degree. C. or 42.degree. C.
during the fermentation to boost the rhPBGD production. However, at
higher fermentation temperatures the strain stability might be a
problem. The time frame was too narrow to study the rhPBGD
expression at all three temperatures, so the decision was to start
the study without temperature induction and to keep the temperature
at 30.degree. C. during the whole process.
[0342] Short Description of the Work
[0343] During the first two months the strain PBGD-1 was cultivated
on agar plates and in shake flasks to obtain information about the
strain characteristics. In parallel to this purchase of study
dedicated chemicals, build up of the documentation system and
technology transfer of the analytical methods took place. When the
PBGD-1 intermediary cell bank was prepared the actual fermentation
work started. First two "simple" 1-L batch fermentations of strain
PBGD-1 were used to test the newly designed substrate and to
calculate the maximum growth rate for the strain. After that three
10 L fed batch fermentations of strain PBGD-1 was performed.
[0344] As soon as the strain PBGD-2 was avaible and an intermediary
cell bank was prepared, this strain was implemented in the
fermentation procedure developed for strain PBGD-1. At present two
10-L fermentations of strain PBGD-2 have been performed.
[0345] The general outline of the fermentation is starting with
inoculum preparation on M9-tc agar plates and shake flasks. The
cells are incubated at 30.degree. C. for 24 h on M9-tc agar plates
and are then transferred to M9-tc shake flasks. The shake flasks
are incubated for 12-14 h at 30.degree. C. The broth from 1-2 shake
flasks are used to inoculate the 10 L fermenter containing a
minimal medium supplemented with yeast extract, trace elements,
thiamine and oxytetracycline as selection pressure. The
fermentation starts with a 14-h batch phase where the cells grow at
maximum growth rate. The glucose feed is started after 14 h and the
feed rate profile is varied between 25-75 ml/h of a 600 gl.sup.-1
glucose solution.
[0346] Broth taken from shake flasks and fermentations have been
used to develop the down stream processing and to test and adjust
the analytical methods provided. The general outline in the down
stream processing is concentration of the fermentation broth on a
0,22 .mu.m cross flow membrane followed by diafiltration (washing)
with a buffer to exchange 90-95% of the substrate with buffer. The
diafiltered cell concentrate is homogenised in a homogeniser, where
the pressure has been varied between 600-1000 bars. The cell debris
is then removed from the homogenate either by filtration on the
same membrane as mentioned above or by centrifugation. Finally the
extract is sterile filtered into sterile containers.
[0347] Results
[0348] Fermentation
[0349] The maximum growth rate for strain PBGD-1 was determined in
shake flask experiments and in 1-L batch fermentations. The results
are summarized in Table 5 below. The reason for the lower values in
the shake flask with fermenter medium is probably acetic acid
production and hence lower pH since the pH is not controlled. No
experiments have been performed to calculate the maximum growth
rate of strain PBGD-2, but from the fact that the batch phase has
the same duration as for PBGD-1 we can draw the conclusion that the
maximum growth rate is approximately the same.
12TABLE 5 Maximum growth rates Maximum growth Conditions rate
(.mu..sub.max) [h.sup.-1] M9-tc Shake flask 0.3 Shake flask with
fermenter substrate 0.3 1 L Fermenter with pH controlled at 7.0
0.4
[0350] The developed substrate for the fermentation is given in
Table 6 on the next page. When implementing strain PBGD-2 it seems
like this strain has different requirements on either the amount of
yeast extract or the thiamine concentration in the substrate. When
using the substrate developed for strain PBGD-1 the growth stops or
lags during the fermentation (PD14). When adding extra yeast
extract and thiamine the growth starts again. This pattern is
repeated at least two times during the fermentation.
13TABLE 6 Fermenter substrate Component Mw [g/mol] Concentration
Unit (NH.sub.4).sub.2SO.sub.4 114.12 2.70 [g/l] KH.sub.2PO.sub.4
136.08 3.25 [g/l] K.sub.2HPO.sub.4 * 3H.sub.2O 228.23 2.80 [g/l]
C.sub.6H.sub.5Na.sub.3O.sub.7 * 2H.sub.2O 258.07 0.60 [g/l] Yeast
extract 5.00-20.0 [g/l] C.sub.6H.sub.12O.sub.6 * H.sub.2O 198.17
10.00 [g/l] MgSO.sub.4 * 7H.sub.2O 246.50 1.07 [g/l] Thiamine
chloride 1.00-10.0 [mg/l] C.sub.12H.sub.18Cl.sub.2N.sub.- 4OS *
xH.sub.2O H.sub.3BO.sub.3 61.83 2.1 [mg/l] CuSO.sub.4 * 5H.sub.2O
249.70 10.5 [mg/l] FeCl.sub.3 * 6H.sub.2O 270.30 35.5 [mg/l]
MnSO.sub.4 * H.sub.2O 169.02 6.6 [mg/l] ZnSO.sub.4 * 7H.sub.2O
287.50 5.3 [mg/l] CoCl.sub.2 * 6H.sub.2O 237.93 9.3 [mg/l]
CaCl.sub.2 * 2H.sub.2O 147.02 14.0 [mg/l] Na.sub.2MoO.sub.4 *
2H.sub.2O 241.95 9.3 [mg/l] HCl 34.46 6.9 [ml/l] Oxytetracycline
496.90 6.0 [mg/l] C.sub.22H.sub.24N.sub.2O.sub.9 * HCl
[0351] The strains seem to utilise different components in the
yeast extract in a sequential order. The metabolism and respiration
is different for different compounds. This gives rise to an
irregular fermentation pattern with large changes in the
respiration of the population during the fermentation, e.g. the
CO.sub.2 and the O.sub.2 outlet gas analysis and the dissolved
oxygen tension (DOT) signal (see FIG. 12).
[0352] As the fermentation proceeds, the fermentation broth is
gradually coloured bright pink. When centrifuging broth for dry
weight analysis it is observed that it is the actual cells and not
the supernatant that is pink. The colonies on the M9-tc agar plates
used to inoculate the shake flasks are not coloured pink, they are
rather yellow or white like "normal" E.coli cells.
[0353] The colonies on the agar plates used for the colony forming
units (CFU) analysis from the fermentation are also pink. However,
on the CFU plates from PD14, the first fermentation with the new
strain PBGD-2, a small portion of yellow or white colonies was
observed. This observation was made already from the plates spread
with broth from the inoculum shake flask. The percentage of
yellow-white cells was varying in the range 2-8% during the
fermentation. Both the white and red colonies were resistant
against the antibiotic oxytetracycline. When observing the white
and red colonies in the microscope they both appeared as E.coli rod
like cells. It was hard to see any clear difference, but possibly
the white cells were a little bit shorter than the red ones. To
investigate this further shake flask cultivation were started with
one red and one yellow colony. The CFU analysis showed that there
were only red colonies from the shake flask inoculated with the red
colony, but that the white colony gave rise to approximately 70%
white and 30% red cells. The rhPBGD activity and protein
concentration were measured in the broth from these shake flasks.
The results are shown in Table 7 below. The difference in the
protein concentration and the rhPBGD activity is in accordance with
the difference in the OD.sub.620 reached in the shake flask,
probably due to different size of the inoculum colony.
14TABLE 7 rhPBGD activity and total protein from single colony
shake flasks Protein PBGD activity Specific activity Start colony
[mg/ml broth] [U/L broth] [U/mg protein] White 0.01 9 0.8 Red 0.04
27 0.7
[0354] In the Table 8 below a summary of the final values of the
fermentations are given. The lower OD.sub.620 and Dw (dry weight)
values in fermentation PD12 is a result of the lower amount of
glucose that totally was fed into the fermenter in this
fermentation (600 ml compared to approximately 850 ml in PD11 and
PD14). It is also interesting to notice the very high expression
and specific activity of rhPBGD in fermentation PD14 compared to
the earlier fermentations.
15TABLE 8 Summary of final fermentation results Specific PBGD
activity Time PBGD activity [U/mg Batch Strain [h] OD.sub.620 Dw
[g/l] [U/ml broth] protein] PD11 PBGD-1 27 82 29 7.7 2.6 PD12
PBGD-1 31 59 19 15.3 1.8 PD14 PBGD-2 30 87 32 39 3.1
[0355] Until now we have achieved the best fermentation results in
fermentation PD14. In the FIGS. 12 and 13 the fermentation results
from this fermentation with the new strain PBGD-2 are shown. After
a 14-h batch phase the glucose feed is started according to a
schedule with three step changes in the feed rate. However after 16
h the glucose begins to accumulate in the fermenter due to that
something else is limiting the growth more. The glucose feed is
then stopped and restarted when the glucose concentration becomes
limiting again.
[0356] The respiration pattern (i.e. CO.sub.2, O.sub.2 and DOT
signals) indicated that something in the substrate was depleted
after 14,5 and 22,3 h and 26,3 h (see FIG. 12). When extra yeast
extract and thiamine was added to the fermenter growth respiration
increased dramatically for a while. There was a steady increase in
the OD.sub.620 and Dw during the whole fermentation and the final
values are rather high. The increase in produced amount of rhPBGD
correlates very well with the increase in biomass. This is
something that has been observed also in the other fermentations.
However, in fermentation PD14 there also seems to be a steady
increase in the specific activity of the produced rhPBGD. Something
that has been much less pronounced in the other fermentations.
[0357] Down Stream Processing
[0358] The different broths have been concentrated 1,9-6,9 times.
The different values reflect problems with clogging of the
membrane. This problem can probably be avoided by not concentrating
the broth too much. Instead a somewhat longer diafiltration has to
be done. The homogenisation has given a good yield of released
enzyme compared to sonication. Removal of cell debris is in the
laboratory scale rather easily done by centrifugation. For the
production scale it would be preferred to use membrane filtration
and because of that filtration has been tested. However, so far the
transmission of enzyme through the membrane has been low resulting
in low yields. This yield may be improved by better controlled
filtration parameters or extended diafiltration. Otherwise a
separator could be used in the production.
[0359] In Table 9 some data from the down stream processing are
shown.
16TABLE 9 Summary of down stream results Sterile filtered extract
Spec. activ- activity Debris protein ity U/mg Yield from broth, %
Broth removal by mg/ml U/ml protein protein U PD11 Filtration 2.7
5.1 1.9 30-60 1) 35-45 1) PD12 Centrifugation 31 84 2.7 79 120 2)
PD14 Centrifugation 32 92 2.9 85 67 PD14 Filtration 3.8 11 2.8 15
18 1) Uncertainties in analysis, because the methods were not fully
evaluated at this time. 2) Uncertainty in volume because of a tube
leakage.
[0360] Conclusions
[0361] Strain PBGD-2 has a maximum growth rate of approximately 0,4
h.sup.-1 in the fermenter substrate. This is similar to the maximum
growth rate of strain PBGD-1, however the substrate requirement
seems to be different for strain PBGD-2. An increase of the initial
yeast extract and thiamine concentration in the substrate to 20
gl.sup.-1 and 10 mgl.sup.-1 respectively supports growth to a
biomass similar to those achieved with the old strain PBGD-1.
[0362] The general fermentation process outline is a 14 h batch
phase followed by a 16 h feed phase were the glucose feed rate is
increased in three steps.
[0363] The production of rhPBGD correlates very well with the
biomass production and the specific activity of the rhPBGD also
seems to increase during the fermentation. The best result so far
with strain PBGD-2 is a rhPBGD concentration of 39 U/ml and a
specific activity of 3,1 U/mg protein after 30-hour fermentation.
The final dry weight and OD.sub.620was 32 gl.sup.-1 and 87
respectively. The plasmid stability is good during the fermentation
when oxytetracycline is present as selection pressure.
EXAMPLE 2
[0364] Development of a Purification Process for Recombinant Human
Porphobilinoqen Deaminase (rhPBGD)
[0365] Introduction
[0366] For the capture step a weak anion exchange (DEAE-Sepharose
FF) matrix has been tested primarily, because this has been the
most common initial step for purification of PBGD. The disadvantage
with anion exchange is that endotoxins and DNA adsorb on this type
of gel. There is a risk that these impurities are coeluted with
rhPBGD. To use a cation exchange in this project is not possible,
because pI for rhPBGD is too low. For that reason a hydrophobic gel
has also been tested as a capture step.
[0367] Material and Methods
[0368] Cell Extract
[0369] Cell extract (PD12, see Example 1) was supplied frozen
(8.times.50 ml) from Biogaia. After the initial thawing a
precipitation was found in the sample. The extract was centrifuged
and the next day a new precipitation was found. This means that the
extract has to be centrifuged in connection to a chromatography
experiment. The protein content in the extract (BCA) was estimated
to 29 mg/ml and the enzyme activity was found to be 63 U/ml. The pH
and conductivity were estimated to 7.0 and 6 mS/cm,
respectively.
[0370] Ion-Exchange
[0371] A DEAE-Sepharose FF hitrap (1 ml) column was used. The gel
was equilibrated with Tris-HCl 25 mM, pH 8.5. The pH of the extract
was adjusted to pH 8.5 with NaOH 5 M and the sample volume applied
on the gel was 1.4 or 2.0 ml. After the sample has been applied,
the gel was washed with 15 column volumes with equilibration
buffer. For the desorption of the gel the following KCl
concentrations have been tested: 40,120, 150 and 300 mM. Finally,
after every experiment the gel was cleaned with NaOH 1 M.
[0372] Hydrophobic Interaction Chromatography
[0373] A Butyl-Sepharose 4 FF hitrap (1 ml) column was used. The
gel was equilibrated with potassium phosphate 1.0-1.3 M pH 7.5. To
the extract, potassium phosphate (2.5 M) was added to an end
concentration of 1.0-1.3 M and the sample volume applied on the gel
was 2.0 ml. After the sample has been applied, the gel was washed
with 15 column volumes with equilibration buffer. For the
desorption of the gel 500 mM, 20 mM potassium phosphate and water
were tested. Finally, after every experiment the gel was cleaned
with NaOH 1 M.
[0374] Results
[0375] Ion-Exchange
[0376] In FIGS. 14 and 15 chromatograms from two DEAE runs are
shown. In Table 10 the results from these runs are shown. The
difference between these experiments are that peak b in the first
run was desorbed with 120 mM KCl and 150 mM in the second. Further,
in the first run less sample was applied and the gel was also
desorbed with 300 mM KCl. The recovery was in the best experiment
found to be 75% and the yield 47%.
[0377] To get this recovery and yield 300 mM KCl has to be used.
The purity of rhPBGD in peak b (DEAE2 ) was estimated to 31%
(RPC).
[0378] Hydrophobic Interaction Chromatography
[0379] In FIG. 16 a chromatogram from a Butyl run is shown. In
Table 11 the result from the run is shown. In this experiment 1.3 M
potassium phosphate was used and the desorption was done with
water. Conductivity in peak b was found to be 60 mS/cm. The
recovery was calculated to 78% and the yield 75%. In an
investigation it was found that a precipitation was formed in the
extract at a potassium phosphate concentration of 1.5 M. The purity
of rhPBGD in peak b was estimated to 40% (RPC).
[0380] Comments and Conclusions
[0381] From the results of the experiments it can be seen that the
mass balance in all experiments are not in balance. This seems to
be valid for all analyses. The main reasons for this are probably
insecurity of the analyses and that all proteins are not eluted
from the gel. The first reason is confirmed by the enzyme activity
that seems to be too high in the extract when high concentration of
potassium phosphate is added. The second reason is confirmed by the
elution peak with NaOH in ion-exchange experiments. This peak is
not analyzed. For the hydrophobic matrix a cleaning with organic
solution can be necessary.
[0382] The conclusion of the results so far is that the
Butyl-Sepharose 4 FF seems to be the best alternative for the
capture step. The main reason for that is the higher yield of
rhPBGD. Another advantage to use Butyl-Sepharose 4 FF is the small
peak after cleaning with NaOH 1M compared with the large peak in
DEAE-Sepharose FF runs. This probably means that few impurities
stick on Butyl matrix. On the other hand there is a risk that a
precipitation is formed when adding potassium phosphate. A
desalting before the next chromatography step can be necessary,
caused by the high ion strength in the product peak.
17TABLE 10 Ion-exchange Applied Peak a Peak b Peak c BCA A280 Act.
BCA A280 Act. BCA A280 Act. BCA A280 Act. mg mg U mg mg U mg mg U
mg mg U Exp. DEAE1 37 166 76 10 122 21 5 6 29 9 11 7 DEAE2 42 229
129 17 143 56 10 14 25 -- -- --
[0383]
18TABLE 11 Hydrophobic interaction chromatography Applied Peak a
Peak b Peak c BCA A280 Act. BCA A280 Act. BCA A280 Act. BCA A280
Act. mg mg U mg mg U mg mg U mg mg U Exp. Butyl 31 137 93 7 84 3 11
23 70 -- -- --
EXAMPLE 3
[0384] Development of a Method for the Purification of Recombinant
Human Porphobilinogen Deaminase with a "His-Tag" (rhPBGD-His)
[0385] Nature and Purpose of the Study
[0386] Many groups have reported in the literature on the
purification of porphobilinogen deaminase from various sources
including E. coli and Human erythrocytes (Anderson P. M. and R. J.
Desnick, 1979, The Journal of Biological Chemistry 255(5): 1993-99,
Awan S. J. et al. 1997, Biochemistry 36(30): 9273-82, Grandchamp B.
et al. 1987, Eur.J.Biochem. 162(1): 105-10, Jordan P. M. 1994,
Wiley, Chichester (Ciba Found Symp 180), p70-96, Jordan P. M. et
al. 1988, Biochhem.J. 254:427-435, Lambert R. et al. Wiley,
Chichester (Ciba Found Symp 180), p97-110, Louie G. V. et al. 1996,
Proteins 25(1): 48-78, Maniatis T., E. F. Fritsch, J. Sambrook.
Molecular Cloning (A laboratory Manual) Cold Spring Harbor
Laboratory. 1982, Miyagi K. et al. 1979, Proc.Natl.Acad.Sci.
76(12):6172-76, Racich N. 1986, Nucleic Acids Research 14(15):
5955-67, Shoolingin-Jordan P. M. et al. 1997, Methods in
Enzymology, 281:317-327). Most use a combination of ion exchange,
hydrophobic interaction and size exclusion chromatography to obtain
fairly pure protein preparations. With the engineering of 5
additional Histidine residues on the C-terminus of recombinant
human porphobilinogen deaminase, rhPBGD we have a convenient "Tag"
to help with purification. Histidine has an affinity to
electropositive transition metals such as nickel, copper, zinc and
cobalt. When a series of 6 or more electron-rich histidine residues
are expressed on the end of a protein they can function as an
anchor, firmly attaching the protein to a solid support coated with
metal ions. Very thorough washing can be done without dislodging
the bound moiety. Elution can be accomplished in one of two ways,
either by decreasing the pH to protonate the imidizole nitrogen
(pKa of 5.97) of histidine, or by including imidizole, a molecule
identical to the histidine side chain, in the elution buffer which
competitively dislodges the tagged protein off the support. The
purpose of this study is to obtain pure rhPBGD-His for antibody
production and for use as a standard in assays and protein
purification.
[0387] Study Objectives
[0388] The objective of this study is to obtain 10 mg of highly
pure active rhPBGD-His.
[0389] Study Plan
[0390] Plan Outline
[0391] 1. Optimize induction time for the expression system and
lysis
[0392] 2. Purify 10 mg of rhPBGD-His for antibody production and
standard
[0393] (2.1) 2 liter scale induction and lysis of strain
[0394] (2.2) DEAE ion exchange chromatography
[0395] (2.3) Immobilized metal affinity chromatography
[0396] 3. Characterization of rhPBGD-His
[0397] (3.1) SDS-PAGE
[0398] (3.2) Amino acid analysis
[0399] (3.3) Specific activity
[0400] (3.4) HPLC
[0401] (3.5) Mass spectrometry
[0402] (3.6) Amino terminal sequencing
[0403] Plan Body
[0404] 1. Expression of rhPBGD-His is regulated by the bacterial
Taq promoter, a derivative of the lac promoter which is inducible
with IPTG (See FIG. 17 for plasmid map). Different proteins are
produced at different rates in E. coli upon induction. This
necessitates the optimization of the time required for optimum
rhPBGD-His yield upon induction. To accomplish this a culture in
mid-log phase will be induced with an excess of IPTG and expression
followed at timepoints with activity and protein concentration
measurements. After induction the cells must be lysed to release
rhPBGD-His. Of the options available, sonication is the best for
this scale of purification. It is compatible with any buffer system
and should not be damaging to the protein. To follow efficiency of
lysis, absorbance at 600 nm will be measured after each cycle.
[0405] 2. For use as a standard and for antibody production at
least 10 mg of rhPBGD-His will be purified.
[0406] (2.1) For protein purification a 2 liter flask culture of
the strain producing rhPBGD-His is sufficient. The culture will be
inoculated with a fresh over-night culture of cells and grown to
mid log phase then induced with IPTG.
[0407] (2.2) Plans are to utilize a two step purification process.
After lysis the debris will be removed by centrifugation and
supernatant loaded onto a DEAE ion exchange column. This will
remove the vast majority of contaminants from the lysate and leave
a limited number of protein contaminants in the elution fractions
containing rhPBGD-His. Protein will be loaded in a high pH and low
ionic strength buffer to ensure binding of the weakly charged
rhPBGD-His. Extensive washing will be used to remove material that
is not firmly bound to the column. A very shallow step gradient of
KCl will be used to elute rhPBGD-His. This should separate the
different forms of rhPBGD-His with differing charge properties from
each other. Separation of different charged forms of PBGD, by ion
exchange chromatography, has been reported by others (Anderson P.
M. and R. J. Desnick, 1979, The Journal of Biological Chemistry
255(5): 1993-99, Jordan P. M. et al. 1988, Biochhem.J. 254:427-435,
Miyagi K. et al. 1979, Proc.Natl.Acad.Sci.USA 76(12):6172-76).
[0408] (2.3) The second chromatographic step planned is a column
containing Talon fast flow immobilized cobalt metal affinity resin
(Clontech). This makes use of the 6-residue histidine tract at the
amino terminus of the recombinant protein. Initially, a metal
chelating resin (Pharmacia) charged with nickel (Sigma) was tried
for purification of rhPBGD-His but it was found to bind other
proteins in the lysate as well which coeluted with rhPBGD-His.
Although this cobalt resin has less binding affinity to His tagged
proteins than nickel it retains the high binding capacity and is
more discriminating which proteins to bind. This leads to elution
from the metal with a lower concentration of imidizole or with
higher pH and achieves a higher level of purity. The cobalt is also
bound more tightly to the matrix by a tetradentate metal chelator,
effectively eliminating the leaching of metal ions from the solid
support during purification. The loss of reactive metal ions during
elution is common problem with nickel based affinity columns
(personal communications) which can lead to unwanted precipitation
of purified proteins.
[0409] 3. rhPBGD-His will be characterized by the following
methods:
[0410] (3.1) The first measure of protein purity will be by
SDS-PAGE (polyacrylamide gel electrophoresis). This method will
also give an indication of the molecular weight of the protein
being produced.
[0411] (3.2) To determine the specific activity of rhPBGD-His in
the preparation it is first necessary to accurately determine
protein concentration in solution. Amino acid analysis will be used
as an accurate method. The method also provides the amino acid
composition of the protein. The concentration can be used to
establish an extinction coefficient of rhPBGD-His.
[0412] (3.3) Activity of the enzyme is an important measure of
correct structure of the enzyme. The proper structure, equivalent
to that produced in humans, is essential for rhPBGD-His to be used
as a therapeutic. Any deviation from the natural structure can
cause activation of the patient's immune system. Historically,
activity of porphobilinogen deaminase has been measured in one of
two ways, either by the metabolism of porphobilinogen substrate or
by the formation of preuroporphyrinogen product. In the reaction
catalyzed by PBGD, porphobilinogen monomers are covalently attached
one at a time starting from the free alpha position of the
dipyrromethane cofactor. After four molecules are added the linear
tetramer of PBG, preuroporphyrinogen, is spontaneously released by
hydrolysis from the cofactor, regenerating the active holoenzyme
with covalently attached cofactor for further reactions (See FIG.
18). After release the tetrapyrrole is circularized by the next
enzyme of the heme pathway, uroporphyrinogen III synthase, forming
uroporphyrinogen III, the central ring of heme and vitamin B12 in
animals and chlorophyll in plants. The linear preuroporphyrinogen
molecule can instead be oxidized to uroporphyrin with benzoquinone
creating a molecule, which absorbs light at 405 nm. This is the
basis for the activity assay used to measure PBGD activity
[0413] (3.4) The rhPBGD-His preparation will be further
characterized by mass spectrometry, which will give an accurate
measure of rhPBGD-His molecular weight and potentially identify
molecular heterogeneity in the preparation. rhPBGD-His can exist
with 1,2,3 and 4 substrate molecules bound to it. Each substrate
molecule added to the holoenzyme will add roughly 209 daltons to
the mass, which is detectable through mass spectrometry.
[0414] (3.5) Characterization by reversed phase HPLC will provide
purity data.
[0415] (3.6) Amino terminal sequencing of rhPBGD-His will be used
to ensure the correct amino terminus.
[0416] Materials and Methods
[0417] Induction and Lysis:
[0418] From a freshly streaked colony, a culture of pExp-2 in JM105
was grown for 13.5 hours in 100 ml LB (10 g/l bacto-tryptone, 5 g/l
bacto-yeast extract, 10 g/l NaCl pH 7.0) +100 .mu.g/ml ampicillin
in a 500 ml baffled flask at 37.degree. C. at 350 rpm. The optical
density measured at 600 nm reached 1.6. This culture was used to
inoculate 2 liters of terrific broth (12 g/l bacto-tryptone, 24 g/l
bacto-yeast extract, 4ml/l glycerol, 2.31 g/l KH2PO4, 12.54 g/l
K.sub.2HPO.sub.4 (Maniatis T., E. F. Fritsch, J. Sambrook.
Molecular Cloning (A laboratory Manual) Cold Spring Harbor
Laboratory. 1982) with 100 .mu.g/ml ampicillin and split into four
2 liter baffled flasks with 400 ml each and two 1 liter baffled
flasks with 200 ml culture each. These were grown at 37.degree. C.
with 350 rpm in a New Brunswick Scientific Innova 4000 incubator.
When reaching an optical density of 0.7 at 600 nm the Taq promoter
was induced with 4 mM IPTG, causing rhPBGD-His protein to be made.
Growth was followed by hourly readings of absorbance at 600 nm.
After 9 hours the cultures were stopped by chilling to 0.degree. C.
after an absorbance of 1.93 was reached. The culture was
centrifuged, 4.times.250 ml at a time, for 10 min at 4,000.times.g
in a Beckman Avanti J251 centrifuge with a JLA-16.250 rotor.
Supernatant was decanted and the remainder of the culture was added
to the cell pellets and spun for an additional 10 min. The pellets
were resuspended in 2 pools of 250 ml 50 mM Tris/HCl pH 8.5
(prechilled) each and stored for 8 hours on ice. Cells were
centrifuged for 10 min at 4,000.times.g, liquid decanted and
resulting pellets weighed in the bottles to determine the wet
weights. Cells were then resuspended in 400 ml ice cold 50 mM
Tris/HCl pH 8.5 and lysed by sonication with a Branson Sonifier 450
with 1/2 inch diameter stepped, tapped horn. Each round was for 30
seconds at maximal power with constant duty cycle in a Pyrex 150 ml
glass beaker on ice. Lysate was mixed between cycles by either
drawing into a 50 ml pipet a few times or by pouring between
beakers on ice. Progress of lysis during sonication was ascertained
by reading absorbance of the lysate at 600 nm. After six rounds of
sonication for each of the 100 ml aliquots of cells, debris was
removed by centrifuging at 16,000.times.g for 30 minutes at
4.degree. C. Lysate was then pooled and vacuum filtered through a
0.22 .mu.m Durapore membrane (Millipore) to remove any remaining
particulate matter.
[0419] DEAE Sepharose Chromatography:
[0420] The first chromatographic step in purifying rhPBGD-His was
by ion exchange chromatography on a DEAE Sepharose fast flow column
(Pharmacia). A 2.5.times.50 cm Spectrum LC column with degassed
resin was washed extensively with degassed 25 mM Tris/HCl pH 8.5
buffer. Filtered lysate (380 ml) was applied to the column at 5
ml/min. The column was then washed with 720 ml 25 mM Tris/HCl pH
8.5. Elution of bound rhPBGD-His was with a shallow step gradient
of KCl from 50 to 120 mM in 10 mM increments in 25 mM Tris/HCl pH
8.5 and degassed. Volumes for each step varied from between 105 and
470 ml depending on the elution profile (see Table 12).
19 TABLE 12 0 mM KCl 50 60 70 80 90 100 110 120 720 ml 470 120 175
270 105 130 180 300
[0421] Fractions were collected about every 50 ml. Absorbance at
280 nm was followed closely during elution. The next step was only
applied after the absorbance had declined following a peak.
BioRad's protein assay II in microtiter format was used per
manufacturer's protocol to assay the amount of protein in each
fraction. Coomassie stained 10% acrylamide Bis/Tris gels (Novex)
were then prepared, with 5 .mu.g protein in each lane, to
characterize the purity of each peak.
[0422] Cobalt Affinity Chromatography:
[0423] The resin slurry was degassed prior to pouring into a
2.5.times.30 cm Spectrum LC column. It was then washed extensively
with degassed 25 mM Tris/HCl pH 8.5/150 mM NaCl at a flow rate of 5
ml/min. The sodium chloride was included to decrease protein to
protein ionic interactions and to reduce ion exchange effects with
the column matrix itself. A relatively high pH of 8.5 was used to
keep rhPBGD-His well above the pI, and therefore negatively
charged, to maintain high solubility during the purification. Two
consecutive rhPBGD-His affinity purifications were then run on the
column. The first sample loaded was a sterile filtered pool of the
entire first peak of eluate of activity from the DEAE sepharose
column including fractions 9 through 12. The column was then washed
with 2 liters of 25 mM Tris pH8.5/150 mM NaCl at 3 ml/min. To elute
bound contaminants the column was then washed with 100 ml of 25 mM
Tris pH8.5/150 mM NaCl/5 mM imidizole at 5 ml/min followed by 100
ml of 10 mM imidizole buffer solution. Elution of his tagged
protein was with 25 mM Tris pH 8.5/150 mM NaCl/50 mM imidizole at 5
ml/min. A final elution with 100 mM imidizole was included to be
certain all rhPBGD-His was eluted. To prepare the column for the
second loading it was merely washed with .about.100 ml of 25 mM
Tris pH8.5/150 mM NaCl. It was hoped that rhPBGD-His would displace
the imidizole bound to the column (which turned out to be the
case). The second loading of the column was with a sterile filtered
pool with .about.900 ml of all remaining peaks of activity from the
DEAE Sepharose column at a flow rate of 5 ml/min. The column was
then washed with 2 liters of 25 mM Tris pH8.5/150 mM NaCl at 5
ml/min, followed by imidizole containing buffers as with the first
run above.
[0424] Polyacrylamide Gel Electrophoresis: (SDS-PAGE)
[0425] Gel electrophoresis was with the Novex system with Nupage
10% Bis/Tris gels run at 125V for 2 hours with or without reducing
agent. Staining was with 50% methanol/10% acetic acid/0.25%
Coomassie brilliant blue R-250 for 2 to 4 hours. Destaining was in
30% methanol/10% acetic acid in a Bio-Rad gel destainer.
[0426] Amino Acid Analysis:
[0427] Amino acid analysis was performed by AAA Laboratory (6206
89.sup.th Avenue Southeast, Mercer Island, Wash. 98040). rhPBGD-His
was hydrolyzed for 20 hours with 6N-HCl/0.05% mercaptoethanol/0.02%
phenol at 11 5.degree. C. Serine was increased by 10% and Threonine
increased by 5% to compensate for destruction of the individual
acids during hydrolysis. A Beckman 7300 Amino Acid Analyzer was
used coupled with System Gold software. Analysis was performed by
post-column derivitization with ninhydrin using the ion-exchange
chromatographic methods developed by Moore and Stein.
[0428] PBGD Activity Assay:
[0429] We performed assays in 96 well microtiter format with
validation in cuvets. Procedures were derived from published
procedures (Awan S. J. et al. 1997, Biochemistry 36(30): 9273-82,
Shoolingin-Jordan P. M. et al. 1997, Methods in Enzymology,
281:317-327). From 0.125 to 8 .mu.g of purified rhPBGD-His protein
per well have been used to determine enzymatic activity. Assay
buffer is 50 mM Tris/HCl pH 8.2 with 1.0 mg/ml BSA (Sigma fraction
5) and 10 mM DTT. A Perkin Elmer 9700 PCR machine was used for
thermal regulation, allowing for tight control of the temperature
and reaction time. Assays have been started in two ways. One method
was to start the reactions at 37.degree. C. with prewarmed
substrate in a PCR block. Strategic placement of pauses in a
thermocycle program was used with beeping at defined intervals for
both addition of the substrate and for stopping the reaction. An
example cycle program is shown in Table 13 with reaction times
varying from 10, 20, 40 and 60 minutes.
[0430] The reaction block is a 96 well block with tubes arranged in
an 8.times.12 matrix. It is kept throughout at 37C. The reaction is
initiated by adding PBG to eight tubes in the first row using an
eight-channel pipettor. The addition is staggered so that each row
receives PBG every 30 seconds. A ten second pause and beep interval
is setup every 20 seconds to signal each addition at the end of the
period. In this fashion all the 96 reactions are started which
takes a total of six minutes. At the end of a further four-minute
incubation, the first three rows are stopped in a staggered manner
giving a total of a ten-minute incubation period. This procedure is
repeated for the next three rows after an additional ten minutes
amounting to a total of twenty-minute reaction time. This scheme is
illustrated in Table 13.
[0431] The p@37 represents the 10-second beep period which is
configured in the thermocyclor as a pause plus beep interval.
20TABLE 13 start stop stop stop stop add 10 20 40 60 PBG min min
min min 5
[0432] Reactions were stopped by acidification with
HCl/p-benzoquinone solution. The final concentration of HCl used
was 1 molar. Benzoquinone, which oxidizes the uroporphyrinogen to
uroporphyrin, was used at a final concentration of 0.002% w/v (from
0.2% stock solution in methanol). At defined intervals the 150
.mu.l samples were removed from the reaction tubes and added to 850
.mu.l HCl/p-benzoquinone solution in wells of a 96 well.times.2 ml
plate on ice. The second method of initiating the assay was to set
up the reactions complete with substrate on ice then to transfer to
the PCR block for incubation at 37.degree. C. Following the
reaction the block was brought to 4.degree. C. to stop the reaction
after which samples were removed and added to HCl/p-benzoquinone
solution. For both methods the incubation was allowed to proceed
for 20 minutes on ice and in the dark after the last addition of
reaction solutions. Then the plate was centrifuged for 10 min at
3750 rpm in a swing out rotor in a GS-6KR centrifuge to pellet
precipitated protein (mostly BSA). 250 .mu.l was removed to a
Corning 96 well assay plate. Absorbance was measured at 405 nm with
a 605 nm reference wavelength in a BioTek FL-600 plate reader.
Selected samples (normally the standard curve) were diluted
10.times. with 1 M HCl and read in a quartz cuvet in a Beckman
DU640B spectrophotometer at 405.5 nm. A 605 nm reference wavelength
was used to subtract out background absorbance. These measurements
in cuvets produced a conversion factor from 1 cm pathlength reads
to the plate data. Analysis was performed using the KC4 software
included with the plate reader and with excel spreadsheets. An
extinction coefficient of 548 M.sup.-1cm.sup.-1 was used to
quantitate the oxidized reaction product (Shoolingin-Jordan P. M.
et al. 1997, Methods in Enzymology, 281:317-327).
[0433] HPLC:
[0434] HPLC analysis was performed at the University of Washington
Mass Spectrometry Analysis Facility for HPLC. Samples were prepared
free of salts for mass spectrometry analysis by HPLC on a C4 column
and eluted with an increasing gradient of acetonitrile. The
instrument used was an Applied Biosystems (ABI) 140A Solvent
Delivery System with an ABI 785A Programmable Absorbance
Detector.
[0435] Mass Spectrometry:
[0436] Mass spectrometric analysis was performed at the University
of Washington Mass Spectrometry Analysis Facility. One tenth of the
HPLC run within the main elution peak was diverted prior to the
absorbance detector to a Perkin Elmer SCIEX API3 Biomolecular Mass
Analyzer for elecro-spray mass spectrometry. Analysis was by
HyperMass method on an average of 16 peaks (for Cobalt run #1
eluate).
[0437] Amino Terminal Sequencing
[0438] Amino terminal sequence analysis was performed at the
University of Washington Mass Spectrometry Analysis Facility. An
ABI 477A Protein Sequencer was used with an ABI 120A PTH
Analyzer.
[0439] Results
[0440] Purification:
[0441] Induction and Lysis:
[0442] Growth of the 2-liter culture of bacteria slowed down after
the first hour but growth still continued to 9 hrs (see Table
14).
21 TABLE 14 start 1 hr 2 hr 3 hr 4 hr 5 hr 6 hr 7 hr 8 hr 9 hr
0.699 1.300 1.521 1.607 1.660 1.732 1.797 1.841 1.890 1.927
[0443] After about 3 hrs of induction cells tended to clump
together with most turbidity settling out of the broth by gravity
in about an hour. Final density of cells stayed low for growth in a
rich media such as terrific broth but final weight of pellets was
adequate. The total wet weight was 35.3 g, corresponding to 17.7
g/liter culture. Interestingly the cells were orange/pink probably
due to various intermediates in the heme biosynthetic pathway. It
is clear from the low growth rate and final densities achieved that
cultures were limited by the amount of oxygen available.
[0444] Lysis by sonication was essentially complete after 5 cycles
as seen by following absorbance readings (Table 15).
22 TABLE 15 # rounds 0 1 2 3 4 5 6 7 OD600 30.33 20.15 12.90 9.43
6.14 3.86 3.31 2.99 % down -- 34 36 27 35 37 14 9.7
[0445] It appears from the % decrease of optical density that for
each of the first 5 rounds of sonication, about the same percentage
of cells were lysed. After this the percentage of newly lysed cells
dropped rapidly. For each of the first 4 rounds viscosity of the
lysate was relatively high due to the presence of unfragmented
genomic DNA but this decreased significantly after further rounds
from shearing of the DNA into smaller fragments.
[0446] DEAE Sepharose:
[0447] Elution of proteins from the DEAE ion exchange column
occurred in 4 distinct peaks as seen in the elution profile in FIG.
19 and by protein assay in Table 16. SDS-PAGE analysis of the
eluted fractions shows that these peaks contain four separate peaks
of rhPBGD-His eluted with the step gradient of KCl (FIG. 20). The
first and major peak was eluted in fractions 9 through 13 with 50
to 70 mM KCl. As seen by gel analysis (see FIG. 20) purity was
fairly good for a first step of the purification, especially in
fractions 10 through 13. The second peak eluted in fractions 15
through 18 with 80 mM KCl. The major contaminant in this peak, in
about equal molar proportions to desired product, was a protein
running at about 5 kDa smaller than rhPBGD-His. The third peak in
fractions 20 through 23 eluted with 90 to 100 mM KCl and had less
visible contaminants than the second peak. The fourth and final
peak eluted in fractions 26 through 29+ with 110 to 120 mM KCl. The
fractions were split into 2 pools for further purification. The
first pool, comprising the major peak of rhPBGD-His elution
contained fractions 9 through 12. The second pool contained
fractions 13 through 29 along with the next 50 ml of 120 mM KCl
elution buffer. These two pools eluted by ion exchange contained
877 mg protein out of 3253 mg loaded, corresponding to a 3.7 fold
decrease in total protein (See Table 16).
[0448] Cobalt Affinity:
[0449] From the first cobalt run the majority of rhPBGD-His eluted
in a sharp peak with a volume of 30 ml upon addition of 50 mM
imidizole (see Table 17 for protein assay and FIG. 22 for SDS-PAGE
results). A final elution with 100 mM imidizole released no
detectable protein absorbing at 280 nm. In the second cobalt run
(FIG. 21) surprisingly, the first imidizole wash of 5 mM eluted a
small uncolored peak of absorbance with a volume of about 50 ml.
The second wash with 10 mM imidizole then eluted a larger and
broader orange/pink colored peak of about 150 ml. Further elution
with 50 mM imidizole yielded a large sharp uncolored peak of 23
ml.
[0450] Characterization:
[0451] Amino Acid Analysis:
[0452] Amino acid analysis of 3 of the fractions (Cobalt run #1 50
mM imidizole eluate (in duplicate,) Cobalt run #2 10 and 50 mM
imidizole eluates) yielded conclusive data that rhPBGD-His was
being purified. Results from the analysis allowed for a very
accurate measure of protein concentration calculated from the
concentration of individual amino acids (see Table 18).
[0453] Specific Activity:
[0454] Specific activity of the first 50 mM imidizole eluate of
rhPBGD-His from the cobalt column turns out to be high at
approximately 24 U/mg (Units are in .mu.mol PBG consumed per mg
protein in one hour). Activity of rhPBGD-His was found to be
strongly dependent on pH with a sharp rise from 7.0 to 8.0 where it
approached a plateau. The optimum was around pH 8.2. The optimum
PBG substrate concentration was found to be around 1 mM. rhPBGD-His
had activity with all concentrations of PBG, however with amounts
less than 1 mM the reaction was limited by available substrate,
decreasing both the Vmax and the linearity over time as substrate
was depleted. It was not found to be necessary to decolorize
remaining benzoquinone with sodium metabisulfite as used in a
published assay (Shoolingin-Jordan P. M. et al. 1997, Methods in
Enzymology, 281:317-327). Strangely enough if acidification and
oxidation were done in a smaller total volume (240 .mu.l vs 1 ml)
as done by this research group then a highly colored product
develops during the incubation on ice. This product must be
decolorized with a saturated solution of sodium metabisulfite to
obtain accurate reaction absorbances. There was no significant
difference in enzymatic activity found as measured by these two
variations of the method.
[0455] Generally assays have been set up with both time and enzyme
concentration as variables. This allows for a more detailed
analysis of the results with a built in validation. If activity is
fairly linear from different timepoints at any enzyme concentration
then it can be inferred that substrate is not limiting and that
reaction measurements are valid over that range. If a measurement
is taken at only one timepoint then there is no indication of
whether the enzyme is still functioning at V-max.
[0456] Reaction volumes in 96 well format have been limited by the
size of PCR tubes to 150 .mu.. Volumes from between 50 and 150
.mu.l have been tried with a noticeable increase in linearity over
time and with increasing enzyme amounts seen with the larger
volumes. Additional increases in volume would make even more
substrate available and dilute the protein further, thereby
increasing the linearity over time and enzyme concentrations.
However the increase in cost of the assay from PBG substrate would
be substantial.
[0457] For routine analysis of similar protein preparations at
similar concentrations it should be possible to standardize the
assay and use far fewer data points and still obtain an accurate
measure of PBGD activity. Optimally a standard curve of rhPBGD-His
of known activity would be included to validate the results and to
simplify analysis. Basically multiple variables including time
would be internally controlled. With a four-parameter logistic
curve of the standards one could use any time point and a wide
range of sample concentrations to obtain accurate activity
measurements. Single use aliquots of highly pure rhPBGD-His could
be stored frozen for use as standards.
[0458] Mass Spectrometry:
[0459] Mass spectrometry of the 50 mM imidizole elution peaks from
the two cobalt runs yielded molecular weights of:
[0460] 1.sup.st cobalt run eluate: 38,816.8, standard
deviation=3.68
[0461] 2.sup.nd cobalt run eluate: 38,814.6, standard
deviation=4.70
[0462] 1.sup.st cobalt eluate dialyzed for antibodies: 38,817.1,
STD deviation=3.33
[0463] These weights correspond to the holoenzyme without any
additional substrate molecules attached.
[0464] Evaluation and Conclusions
[0465] We found that with a simple two step purification process
involving ion exchange and cobalt affinity chromatography we could
achieve a yield of 173 mg/l rhPBGD-His with a purity of greater
than 98% starting from a bacterial crude lysate. Each one of the
enzyme intermediate complexes is stable and can be independently
isolated (Anderson P. M. and R. J. Desnick, 1979, The Journal of
Biological Chemistry 255(5): 1993-99, Jordan P. M. et al. 1988,
Biochhem.J. 254:427-435, Miyagi K. et al. 1979,
Proc.Natl.Acad.Sci.USA 76(12):6172-76). This may be a major
contributing factor to the differential binding of different enzyme
fractions to the DEAE ion exchange matrix. Due to the negative
charges contributed by acetate and propionate side groups on the
growing chain of porphobilinogen molecules it could be theorized
that binding affinity to the ion exchange resin would be in the
order; E<ES<ES2<ES3. That would imply that the first peak
could be the holoenzyme followed by the others in the same order of
the reaction progression. The cobalt column also eluted rhPBGD-His
in different fractions during the second run. It is strange that
the elution profile from the second run was different from the
first. It would be expected that all closely related proteins with
a his-tag would bind to cobalt with the same affinity. This implies
that either the His-tag is partially digested away or partially
obscured due to protein conformational changes or charge
interactions. The difference in elution characteristics may also be
due to differences between the various enzyme-substrate
intermediate complexes as hinted by the color difference in the 10
mM elution peak. From a report in the literature by Jordan P. M.
1994, Wiley, Chichester (Ciba Found Symp 180), p70-96, the ES2
intermediate complex has a pink colored chromophore. The 10 mM
imidizole fraction from the second cobalt column run has a pink
color while the other fractions do not. This implies a separation
of different enzyme substrate intermediates in different fractions.
If the colored protein peak is predominantly composed of the ES2
intermediate then it could be extrapolated that the peak at 5 mM
would be ES3. Whatever would be decreasing the binding of ES2 to
cobalt whether conformational or charge related as compared to ES
would likely be enhanced with the ES3 intermediate. The peak
released with 50 mM imidizole and with stronger binding to DEAE
could then be the ES form. Holoenzyme by itself may be the
remaining form, purified during the first cobalt run, binding less
tightly to DEAE due to a higher .mu.l and elute from nickel with 50
mM imidizole. When the mass of the two 50 mM cobalt eluates were
compared however there was no significant difference detected. Both
corresponded to the weight expected for holoenzyme alone.
Unfortunately no reliable mass measurement of the 10 mM eluate was
obtained due possibly to precipitation problems with a lower
rhPBGD-His protein concentration. If a difference of elution
characteristics between the different enzyme substrate
intermediates is occurring then a likely explanation would be due
to the large conformational changes that take place during the
course of the reactions From E to ES4 (Jordan P. M. 1994, Wiley,
Chichester (Ciba Found Symp 180), p70-96, Louie G. V. et al. 1996,
Proteins 25(1): 48-78). The C-terminal His-tag on the third domain
of the protein could become partially hidden and rendered
sterically less accessible when the reaction proceeds past the ES1
form. A direct interaction between the his-tag and the growing
substrate chain would be less likely. At a pH of 8.5 histidines
should be in an electron rich unprotonated state and the substrate
complex should also be in an electron rich state even though acidic
side chains are neutralized by basic amino acids in the catalytic
cleft (Jordan P. M. 1994, Wiley, Chichester (Ciba Found Symp 180),
p70-96, Louie G. V. et al. 1996, Proteins 25(1): 48-78).
Conformational changes in rhPBGD-His occurring during the reaction
could conceivably make accessible other charge groups for
interaction with the his-tag either on the surface or perhaps the
same ones meant for dampening charges from the growing substrate
polymer in the cleft.
[0466] Equipment and supplies lists are shown in appendix 4 and 5,
respectively.
23TABLE 16 Protein assay results on DEAE fractions (with BioRad's
Protein assay II kit): protein rhPBGD-His protein sample vol(ml) mM
KCl mg/ml mg/ml mg prot gel # well # pool mg pool DEAE Load 380 0
8.56 3253 1 2 DEAE FT 380 0 1.33 505 1 3 DEAE #1 16 0 0.26 0.00 4 1
4 2 60 0 0.23 0.00 14 1 5 3 100 0 0.044 0.00 4 1 6 4 215 0 0.002
0.00 0 1 7 5 320 0 0.005 0.00 2 1 8 6 75 50 0.11 0.00 8 1 9 7 100
50 0.19 0.00 19 1 10 8 100 50 0.14 0.00 14 1 11 9 94 50 0.61 0.31
57 1 12 1 334 10 100 50 1.44 1.15 144 2 1 1 I 11 38 60 1.05 0.84 40
2 2 1 I 12 135 60-70 0.69 0.48 93 2 3 1 V 13 100 70 0.4 0.10 40 2 4
2 543 14 50 80 0.43 0.09 22 2 5 2 I 15 50 80 0.96 0.34 48 2 6 2 I
16 50 80 0.98 0.49 49 2 7 2 I 17 50 80 0.57 0.29 29 2 8 2 I 18 50
80-90 0.48 0.14 24 2 9 2 I 19 50 90 0.42 0.13 21 2 10 2 I 20 50 90
0.61 0.24 31 3 1 2 I 21 50 90-100 0.93 0.70 47 3 2 2 I 22 50 100
1.08 0.86 54 3 3 2 I 23 50 100 0.57 0.34 29 3 4 2 I 24 50 110 0.41
0.08 21 3 5 2 I 25 50 110 0.61 0.09 31 3 6 2 I 26 50 110 0.69 0.17
35 3 7 2 I 27 50 110-120 0.73 0.44 37 3 8 2 I 28 28 120 1.07 0.86
30 3 9 2 I 29 50 120 1.08 0.76 54 3 10 2 V
[0467]
24TABLE 17 Second cobalt run fractions with other samples in FIG.
20 gel Sample Imidizole volume Conc. Total Gel Ico-pure description
mM ml mg/ml mg lane mg Co-1 load 0 367 0.78 286.3 2 Co-1 FT 0 367
0.22 80.7 3 Co-1 Eluate 50 30 6.46 193.8 4 193.8 MI #1 Nickel 500 ?
9.65 5 PBGD-1 Lys 0 1.73 6 Ab prep #1 0 3.3 4.00 13.2 7 Cobalt-2 FT
0 900 0.23 207.0 8 FT tail 0 30 0.02 0.6 -- Cobalt-2 w1 5 100 0.01
1.0 -- Cobalt-2 w2 5 50 0.14 7.0 -- Cobalt-2 w3 5 52 0.02 1.0 --
Cobalt-2 w4 5 100 0.04 4.0 -- Cobalt-2 w5 10 50 0.07 3.5 --
Cobalt-2 w6 10 50 0.81 40.5 9 101.5 Cobalt-2 w7 10 50 0.81 40.5 10
Cobalt-2 w8 10 50 0.41 20.5 11 Cobalt-2 w9 10 130 0.20 26.0 --
Cobalt-2 E1 50 22.5 2.29 51.5 12 51.5 Cobalt-2 E2 50 30 0.11 3.3 --
Total mg highly pure rhPBGD-His (by amino acid analysis) =
346.8
[0468]
25TABLE 18 rhPBGD-His amino acid analysis: Cobalt eluates #1 #2 #3
#4 umol/ml aa #aa % whole aa [PBGD] aa [PBGD] aa [PBGD] aa [PBGD]
Ala 29 8.31 4.8220 0.166 0.5920 0.020 1.6950 0.058 4.5406 0.157 Arg
21 6.02 3.7279 0.178 0.4550 0.022 1.3050 0.062 3.5129 0.167 Asn 10
2.87 Asp 19 5.44 4.7157 0.163 0.5755 0.020 1.6510 0.056 4.4442
0.153 Cys 4 1.15 Gln 19 5.44 Glu 21 6.02 6.7648 0.169 0.8152 0.020
2.3592 0.059 6.3524 0.159 Gly 27 7.74 4.3933 0.163 0.6120 0.023
1.5595 0.058 4.1137 0.152 His 18 5.16 2.5664 0.143 0.2539 0.014
0.7947 0.044 2.4518 0.136 Ile 20 5.73 3.0511 0.153 0.3661 0.018
1.0561 0.053 2.8381 0.142 Leu 43 12.32 7.0235 0.163 0.8565 0.020
2.4506 0.057 6.6020 0.154 Lys 18 5.16 2.9241 0.162 0.3538 0.020
1.0427 0.058 2.7135 0.151 Met 6 1.72 0.8691 0.145 0.0996 0.017
0.3072 0.051 0.8252 0.138 Phe 9 2.58 1.4713 0.163 0.1825 0.020
0.5216 0.058 1.4045 0.156 Pro 16 4.58 2.7268 0.170 0.3708 0.023
1.1194 0.070 2.8441 0.178 Ser 18 5.16 2.8356 0.158 0.3570 0.020
1.0121 0.056 2.8680 0.159 Thr 20 5.73 3.4426 0.172 0.4272 0.021
1.2154 0.061 3.2746 0.164 Trp 2 0.57 Tyr 3 0.86 0.5150 0.172 0.0626
0.021 0.1734 0.058 0.4823 0.161 Val 26 7.45 4.0526 0.156 0.4920
0.019 1.4292 0.055 3.7740 0.145 Avg .mu.mol/mol rhPBGD-His: 0.167
0.021 0.059 0.159 (w/o bold values) Avg mg/ml rhPBGD-His: 6.46 0.81
2.29 6.17 (MW = 38759.4) Volume (ml): 30 100 22.5 5 amount in
fraction (mg): 193.7 80.8 51.6 30.8 sample run # imidazole Notes #1
1 50 mM Major peak of cobalt eluate (from 50->60 mM KCl elution
from DEAE) #2 2 10 mM 70->120 mM KCl elution from DEAE; cobalt
fractions W-6,7 #3 2 50 mM 70->120 mM KCl elution from DEAE;
fraction E-1 #4 1 -- #1 dialyzed->PBS + 5 mM Tris/Cl pH 8.0
(used for 2nd round Antibodies) Analysis done at: AAA Laboratory
By: Lowell Ericsson, Nancy Ericsson Address: 6206 89th Ave SE,
Mercer Island, Washington 98040-4599
[0469] Combination Therapy
[0470] Combination Therapy of rhALAD AND rhPBGD
[0471] The etiology behind AIP is not fully understood. However,
the accumulation of the two heme-precursors delta-aminolevulinic
acid (ALA) and porphobilinogen (PBG) are likely to be involved. ALA
and PBG have been suggested to be toxic to the central and
peripheral nervous system causing the well known symptoms such as
abdominal pain, muscle weakness, loss of sensory functions as well
as epileptic seizures, respiratory paralysis, hallucinations and
psychosis, observed during acute attacks.
[0472] The rationale for the enzyme substitution therapy in AIP
patients is based on the administration of rhPBGD by sc. injections
to lower serum and intracellular PBG levels. PBG will be
metabolized to preurophorphyrinogen. Preuroporphyrinogen will
subsequently enter the normal heme biosynthetic pathway and be
metabolized to heme. Hence, rhPBGD enzyme replacement therapy will
have a dual action, i) reduce circulating levels of toxic PBG and
ii) restore heme production.
[0473] In the etiology of the disease it has always been suggested
that ALA might have an even more toxic effect that PBG. Hence, a
reduction of both ALA and PBG is important to achieve. Treatment of
AIP patients with rhPBGD will i) reduce circulating levels of PBG
as well as ALA, since ALA and PBG are in equilibrium with each
other through coupled enzyme reactions e.g. deta-aminolevulinic
acid dehydratase (ALAD) and porphobilinogen deaminase PBGD and ii)
restore heme production. A block in the PBGD enzyme will result in
the accumulation of both PBG and ALA. Administration of rhPBGD will
quickly metabolise PBG and lower ALA levels as well, through
changes in the equilibrium of the ALAD enzyme reaction.
[0474] An accelerated reduction of ALA might be beneficial to AIP
patients. Hence, a coadministration of both rhPBGD and rhALAD will
rapidly reduce both heme precursors. The mixing and administration
of rhALAD and rhPBGD could be done in two ways, either: i) a
product containing both enzymes at fixed proportions or ii)
administration of rhPBGD and rhALAD by two separate subcutaneous
injections. In the latter case the dose of the two enzymes could be
adjusted to obtain optimal individual therapy. Administration of
separate enzymes provides also a possibility for optimal temporal
order of administration to obtain the best individual therapeutic
effect.
[0475] Combination Therapy of rhPBGD and rhuroporphyrinogen III
cosynthetase
[0476] Coadministration of rhPBGD and rhuroporphyrinogen III
cosynthetase to some AIP patients are likely to be beneficial, by
improving conversion of preuroporphyrinogen to its uroporphyrinogen
III isomer rather than the I isomer. The I isomer forms
spontaneously from preuroporphyrinogen and can not be further
metabolised into heme. Hence, a coadministration of rhPBGD and
Uroporphyrinogen III cosynthetase will ensure a better restoration
of normal heme synthesis in that less amount of the
uroporphyrinogen I isomer will be formed.
[0477] Combination Therapy of rhALAD, rhPBGD and
rhUroporphyrinoqenIIIcosy- nthetase
[0478] rhPBGDcosynthetase can be coadminstered with both rhPBGD and
rhALAD to specific patients to obtain beneficial heme synthesis
restoration.
[0479] It is within the scope of the present invention to extend a
combination therapy to other enzymes mentioned herein and to
treatment of the other porphyias.
[0480] Treatment of other Porphyrias
[0481] 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 rhALA dehydratase, rhUroporphyrinogen
decarboxylase, rhCoproporphyrinogen oxidase and
rhProtoporphyrinogen oxidase, respectively.
[0482] Patients having Erythropoetic Porphyrias such as Congenital
erythropoietic Porphyria (CEP) or Erythropoietic protoporphyria
(EPP) will benefit from substitution therapy with
rhUroporphyrinogen III synthetase and rhFerrochelatase,
respectively.
[0483] Hepatoerythropoietic Porphyriase.g. Hepatoerythropoietic
Porphyrias (HEP) can be treated with rhUroporphyrinogen
decarboxylase.
[0484] 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.
[0485] 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. In situations
where a combination of enzymes are beneficial, such therapy may be
applied in a manner similar as disclosed above.
[0486] References:
[0487] Anderson P. M. and R. J. Desnick. 1979, Purification and
Properties of Uroporphyrinogen I Synthase from Human Erythrocytes:
Identification of Stable Enzyme-Substrate Intermediates The Journal
of Biological Chemistry 255(5) :1993-99
[0488] Andersson, Christer, Thesis, 1997, ISBN 91/7191/280/0, pp.
22-23
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bearing a hybrid trp-lac promoter useful for regulated expression
of cloned genes in Escherichia coli. Gene 25(2-3):167-178
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Form of E. coli Porphobilinogen Deaminase from Apoenzyme with
Porphobilinogen and Preuroporphyrinogen: A Study Using Circular
Dichroism Spectroscopy Biochemistry 36 (30) :9273-82
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[0497] Jordan, P. M. The biosynthesis of uroporphyrinogen III:
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Proteins 25(1):48-78
[0502] Makrides, S. C. 1996, Strategies for achieving high-level
expression of genes in Escherichia coli. Microbiol.Rev.
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Sequence CWU 1
1
40 1 5446 DNA Artificial Sequence Sequence of the expression
plasmid pExp1-M2-BB 1 gaattctaac ataagttaag gaggaaaaaa aaatgagagt
tattcgtgtc ggtacccgca 60 agagccagct tgctcgcata cagacggaca
gtgtggtggc aacattgaaa gcctcgtacc 120 ctggcctgca gtttgaaatc
attgctatgt ccaccacagg ggacaagatt cttgatactg 180 cactctctaa
gattggagag aaaagcctgt ttaccaagga gcttgaacat gccctggaga 240
agaatgaagt ggacctggtt gttcactcct tgaaggacct gcccactgtg cttcctcctg
300 gcttcaccat cggagccatc tgcaagcggg aaaaccctca tgatgctgtt
gtctttcacc 360 caaaatttgt tgggaagacc ctagaaaccc tgccagagaa
gagtgtggtg ggaaccagct 420 ccctgcgaag agcagcccag ctgcagagaa
agttcccgca tctggagttc aggagtattc 480 ggggaaacct caacacccgg
cttcggaagc tggacgagca gcaggagttc agtgccatca 540 tcctggcaac
agctggcctg cagcgcatgg gctggcacaa ccgggttggg cagatcctgc 600
accctgagga atgcatgtat gctgtgggcc agggggcctt gggcgtggaa gtgcgagcca
660 aggaccagga catcttggat ctggtgggtg tgctgcacga tcccgagact
ctgcttcgct 720 gcatcgctga aagggccttc ctgaggcacc tggaaggagg
ctgcagtgtg ccagtagccg 780 tgcatacagc tatgaaggat gggcaactgt
acctgactgg aggagtctgg agtctagacg 840 gctcagatag catacaagag
accatgcagg ctaccatcca tgtccctgcc cagcatgaag 900 atggccctga
ggatgaccca cagttggtag gcatcactgc tcgtaacatt ccacgagggc 960
cccagttggc tgcccagaac ttgggcatca gcctggccaa cttgttgctg agcaaaggag
1020 ccaaaaacat cctggatgtt gcacggcaat tgaacgatgc ccattaataa
gcttggctgt 1080 tttggcggat gagagaagat tttcagcctg atacagatta
aatcagaacg cagaagcggt 1140 ctgataaaac agaatttgcc tggcggcagt
agcgcggtgg tcccacctga ccccatgccg 1200 aactcagaag tgaaacgccg
tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta 1260 gggaactgcc
aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt 1320
tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt
1380 gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat
aaactgccag 1440 gcatcaaatt aagcagaagg ccatcctgac ggatggcctt
tttgcgtttc tacaaactct 1500 tttgtttatt tttctaaata cattcaaata
tgtatccgct catgagacaa taaccctgat 1560 aaatgcttca ataatattga
aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 1620 ttattccctt
ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 1680
aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
1740 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
atgagcactt 1800 ttaaagttct gctatgtggc gcggtattat cccgtgttga
cgccgggcaa gagcaactcg 1860 gtcgccgcat acactattct cagaatgact
tggttgagta ctcaccagtc acagaaaagc 1920 atcttacgga tggcatgaca
gtaagagaat tatgcagtgc tgccataacc atgagtgata 1980 acactgcggc
caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 2040
tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
2100 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
acgttgcgca 2160 aactattaac tggcgaacta cttactctag cttcccggca
acaattaata gactggatgg 2220 aggcggataa agttgcagga ccacttctgc
gctcggccct tccggctggc tggtttattg 2280 ctgataaatc tggagccggt
gagcgtgggt ctcgcggtat cattgcagca ctggggccag 2340 atggtaagcc
ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 2400
aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
2460 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
tttaaaagga 2520 tctaggtgaa gatccttttt gataatctca tgaccaaaat
cccttaacgt gagttttcgt 2580 tccactgagc gtcagacccc gtagaaaaga
tcaaaggatc ttcttgagat cctttttttc 2640 tgcgcgtaat ctgctgcttg
caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 2700 cggatcaaga
gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 2760
caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
2820 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
ggcgataagt 2880 cgtgtcttac cgggttggac tcaagacgat agttaccgga
taaggcgcag cggtcgggct 2940 gaacgggggg ttcgtgcaca cagcccagct
tggagcgaac gacctacacc gaactgagat 3000 acctacagcg tgagctatga
gaaagcgcca cgcttcccga agggagaaag gcggacaggt 3060 atccggtaag
cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 3120
cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
3180 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
tttttacggt 3240 tcctggcctt ttgctggcct tttgctcaca tgttctttcc
tgcgttatcc cctgattctg 3300 tggataaccg tattaccgcc tttgagtgag
ctgataccgc tcgccgcagc cgaacgaccg 3360 agcgcagcga gtcagtgagc
gaggaagcgg aagagcgcct gatgcggtat tttctcctta 3420 cgcatctgtg
cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg 3480
ccgcatagtt aagccagtat acactccgct atcgctacag atccggaaca taatggtgca
3540 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
ccattcatgt 3600 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
cacgttcgct cgcgtatcgg 3660 tgattcattc tgctaaccag taaggcaacc
ccgccagcct agccgggtcc tcaacgacag 3720 gagcacgatc atgcgcaccc
gtggccagga cccaacgctg cccgagatgc gccgcgtgcg 3780 gctgctggag
atggcggacg cgatggatat gttctgccaa gggttggttt gcgcattcac 3840
agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt tagcgaggtg
3900 ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg
caacgcgggg 3960 aggcagacaa ggtatagggc ggcgcctaca atccatgcca
acccgttcca tgtgctcgcc 4020 gaggcggcat aaatcgccgt gacgatcagc
ggtccagtga tcgaagttag gctggtaaga 4080 gccgcgagcg atccttgaag
ctgtccctga tggtcgtcat ctacctgcct ggacagcatg 4140 gcctgcaacg
cgggcatccc gatgccgccg gaagcgagaa gaatcataat ggggaaggcc 4200
atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc cgccatgccg
4260 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
gaaggcttga 4320 gcgagggcgt gcaagattcc gaataccgca agcgacaggc
cgatcatcgt cgcgctccag 4380 cgaaagcggt cctcgccgaa aatgacccag
agcgctgccg gcacctgtcc tacgagttgc 4440 atgataaaga agacagtcat
aagtgcggcg acgatagtca tgccccgcgc ccaccggaag 4500 gagctgactg
ggttgaaggc tctcaagggc atcggtcgac gctctccctt atgcgactcc 4560
tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
4620 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
caccataccc 4680 acgccgaaac aagcgctcat gagcccgaag tggcgagccc
gatcttcccc atcggtgatg 4740 tcggcgatat aggcgccagc aaccgcacct
gtggcgccgg tgatgccggc cacgatgcgt 4800 ccggcgtaga ggatccacag
gacgggtgtg gtcgccatga tcgcgtagtc gatagtggct 4860 ccaagtagcg
aagcgagcag gactgggcgg cggccaaagc ggtcggacag tgctccgaga 4920
acgggtgcgc atagaaattg catcaacgca tatagcgcta gcagcacgcc atagtgactg
4980 gcgatgctgt cggaatggac gatatcccgc aagaggcccg gcagtaccgg
cataaccaag 5040 cctatgccta cagcatccag ggtgacggtg ccgaggatga
cgatgagcgc attgttagat 5100 ttcatacacg gtgcctgact gcgttagcaa
tttaactgtg ataaactacc gcattaaagc 5160 taatcgatga taagctgtca
aacatgagtg atccgggctt atcgactgca cggtgcacca 5220 atgcttctgg
cgtcaggcag ccatcggaag ctgtggtatg gctgtgcagg tcgtaaatca 5280
ctgcataatt cgtgtcgctc aaggcgcact cccgttctgg ataatgtttt ttgcgccgac
5340 atcataacgg ttctggcaaa tattctgaaa tgagctgttg acaattaatc
atcggctcgt 5400 ataatgtgtg gaattgtgag cggataacaa tttcacacag gaaaca
5446 2 3225 DNA Artificial Sequence Sequence of the EcoR I - Hind
III linear fragment used for transformation in the hemC disruption
strategy 2 aattcgtcaa gcagcagtat atgctgggtg gagccacaat cttcgccccc
caggctgccg 60 ctttcattat gacggaagcg gttttcatca atcaggaaga
agctgacttc cacacccagc 120 gaggcggccc agttttccag caggctacat
ttacgttgta gcaattggcg ctcttcgcta 180 tcgagccagg attgatgaca
gacccagata tccaggtcag aggaacaact ttgccctacg 240 gacgaggtgc
tgcccatggt gtatacacca gtaattggaa gctcaccttt cggcggatcc 300
tgtactgaca ttccacgata cagttcaagc tcgttcaggt agtggcgttg agtttcatca
360 ggcgtgtaaa ggcaaatgcc tttgggaacg ttaccatcaa ggtagcccgg
cattagcgga 420 tggtgatagt gcaacaatgt cggcagtaga ctgtagacct
gttggaatgc aggccccata 480 gcagcaagcg cgcgatccac acgcaattga
tttatggcat ccagtctctg tttcagagtc 540 tcaatataga ggtacaagac
gtatcgcctg atttgctacc cgtcatgact gtgattccgc 600 caacatcaac
ggtaacacgc ggcattcggg atatttcgta tgtcaaaggt aaccgttacc 660
acttttcgcg cctggttttt ttagtttcac gacgaaaaaa tggtctaaaa cgtgatcaat
720 ttaacacctt gctgattgac cgtaaagaaa gatgcgctac atacaagtgt
agcaccgttt 780 attctctgta aattccttat tacaacggcg tgaaacgcct
gtcaggatcc actgccagac 840 ctcattttac ggtttgcgca ggcgtctacg
tttcaccaca acactgacat cactctggca 900 aggatgttag gatggaccac
ggatgataat gacggtaaca agcatgttag acaatgtttt 960 aagaattgcc
acacgccaaa gcccacttgc actctggcag gcacactatg tcaaagacaa 1020
gttgatggcg agccatccgg gcctggtcgt tgaactggta ccgatggtga cctcgagcgg
1080 cacgtaagag gttccaactt tcaccataat gaaataagat cactaccggg
cgtatttttt 1140 gagttgtcga gattttcagg agctaaggaa gctaaaatgg
agaaaaaaat cactggatat 1200 accaccgttg atatatccca atggcatcgt
aaagaacatt ttgaggcatt tcagtcagtt 1260 gctcaatgta cctataacca
gaccgttcag ctggatatta cggccttttt aaagaccgta 1320 aagaaaaata
agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat 1380
gctcatccgg aattacgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt
1440 cacccttgtt acaccgtttt ccatgagcaa actgaaacgt tttcatcgct
ctggagtgaa 1500 taccacgacg atttccggca gtttctacac atatattcgc
aagatgtggc gtgttacggt 1560 gaaaacctgg cctatttccc taaagggttt
attgagaata tgtttttcgt ctcagccaat 1620 ccctgggtga gtttcaccag
ttttgattta aacgtggcca atatggacaa cttcttcgcc 1680 cccgttttca
ccatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg 1740
attcaggttc atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta
1800 caacagtact gcgatgagtg gcagggcggg gcgtaattct cgagaccggc
atgagtatcc 1860 ttgtcacccg cccgtctccc gctggagaag agttagtgag
ccgtctgcgc acactggggc 1920 aggtggcctg gcattttccg ctgattgagt
tttctccggg tcaacaatta ccgcaacttg 1980 ctgatcaact ggcagcgctg
ggggagagcg atctgttgtt tgccctctcg caacacgcgg 2040 ttgcttttgc
ccaatcacag ctgcatcagc aagatcgtaa atggccccga ctacctgatt 2100
atttcgccat tggacgcacc accgcactgg cactacatac cgtaagtgga cagaagattc
2160 tctacccgca ggatcgggaa atcagcgaag tcttgctaca attacctgaa
ttacaaaata 2220 ttgcgggcaa acgtgcgctg atattacgtg gcaatggtgg
tcgtgagcta attggggata 2280 ccctgacggc gcgcggtgct gaggtcactt
tttgtgaatg ttatcaacga tgcgcaatcc 2340 attacgatgg tgcagaagaa
gcgatgcgct ggcaagcccg cgaggtgacg atggtcgttg 2400 ttaccagcgg
tgaaatgttg cagcaactct ggtcgctgat cccacaatgg tatcgtgagc 2460
actggttact acactgtcga ctattggtcg tcagtgagcg tttggcgaaa ctcgcccggg
2520 aactgggctg gcaagacatt aaggtcgccg ataacgctga caacgatgcg
cttttacggg 2580 cattacaata actctcataa caggaagcca taatgacgga
acaagaaaaa acctccgccg 2640 tggttgaaga gaccagggag gccgtggaca
ccacgtcaca acctgtcgca acagaaaaaa 2700 agagtaagaa caataccgca
ttgattctca gcgcggtggc tatcgctatt gctctggcgg 2760 cgggcatcgg
tttgtatggc tggggtaaac aacaggccgt caatcagacc gccaccagcg 2820
atgccctggc taaccaactg acggcattgc aaaaagccca ggagagccaa aaagccgagc
2880 tggaaggcat tattaagcaa caagctgcac aacttaagca ggcgaatcgt
cagcaagaaa 2940 cgctggcaaa acagttggat gaagtccaac aaaaggtcgc
caccatttcc ggcagcgatg 3000 ctaaaacctg gctgctggct caggccgatt
ttctggtgaa actcgccgga cggaagctgt 3060 ggagcgatca ggacgtcacg
accgctgcag cgttgctgaa aagtgcagac gccagcctgg 3120 cggatatgaa
tgacccgagt ctgattaccg ttcgtcgggc aattaccgat gatatcgcca 3180
gcctttctgc agtatcgcag gtggattatg acggcatcat cctta 3225 3 1035 DNA
Human tissue 3 atgagagtga ttcgcgtggg tacccgcaag agccagcttg
ctcgcataca gacggacagt 60 gtggtggcaa cattgaaagc ctcgtaccct
ggcctgcagt ttgaaatcat tgctatgtcc 120 accacagggg acaagattct
tgatactgca ctctctaaga ttggagagaa aagcctgttt 180 accaaggagc
ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg 240
aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg caagcgggaa
300 aaccctcatg atgctgttgt ctttcaccca aaatttgttg ggaagaccct
agaaaccctg 360 ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag
cagcccagct gcagagaaag 420 ttcccgcatc tggagttcag gagtattcgg
ggaaacctca acacccggct tcggaagctg 480 gacgagcagc aggagttcag
tgccatcatc ctggcaacag ctggcctgca gcgcatgggc 540 tggcacaacc
gggttgggca gatcctgcac cctgaggaat gcatgtatgc tgtgggccag 600
ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct ggtgggtgtg
660 ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa gggccttcct
gaggcacctg 720 gaaggaggct gcagtgtgcc agtagccgtg catacagcta
tgaaggatgg gcaactgtac 780 ctgactggag gagtctggag tctagacggc
tcagatagca tacaagagac catgcaggct 840 accatccatg tccctgccca
gcatgaagat ggccctgagg atgacccaca gttggtaggc 900 atcactgctc
gtaacattcc acgagggccc cagttggctg cccagaactt gggcatcagc 960
ctggccaact tgttgctgag caaaggagcc aaaaacatcc tggatgttgc acggcaattg
1020 aacgatgccc attaa 1035 4 1113 DNA Human tissue 4 cacacagcct
actttccaag cggagccatg tctggtaacg gcaatgcggc tgcaacggcg 60
gaagaaaaca gcccaaagat gagagtgatt cgcgtgggta cccgcaagag ccagcttgct
120 cgcatacaga cggacagtgt ggtggcaaca ttgaaagcct cgtaccctgg
cctgcagttt 180 gaaatcattg ctatgtccac cacaggggac aagattcttg
atactgcact ctctaagatt 240 ggagagaaaa gcctgtttac caaggagctt
gaacatgccc tggagaagaa tgaagtggac 300 ctggttgttc actccttgaa
ggacctgccc actgtgcttc ctcctggctt caccatcgga 360 gccatctgca
agcgggaaaa ccctcatgat gctgttgtct ttcacccaaa atttgttggg 420
aagaccctag aaaccctgcc agagaagagt gtggtgggaa ccagctccct gcgaagagca
480 gcccagctgc agagaaagtt cccgcatctg gagttcagga gtattcgggg
aaacctcaac 540 acccggcttc ggaagctgga cgagcagcag gagttcagtg
ccatcatcct ggcaacagct 600 ggcctgcagc gcatgggctg gcacaaccgg
gttgggcaga tcctgcaccc tgaggaatgc 660 atgtatgctg tgggccaggg
ggccttgggc gtggaagtgc gagccaagga ccaggacatc 720 ttggatctgg
tgggtgtgct gcacgatccc gagactctgc ttcgctgcat cgctgaaagg 780
gccttcctga ggcacctgga aggaggctgc agtgtgccag tagccgtgca tacagctatg
840 aaggatgggc aactgtacct gactggagga gtctggagtc tagacggctc
agatagcata 900 caagagacca tgcaggctac catccatgtc cctgcccagc
atgaagatgg ccctgaggat 960 gacccacagt tggtaggcat cactgctcgt
aacattccac gagggcccca gttggctgcc 1020 cagaacttgg gcatcagcct
ggccaacttg ttgctgagca aaggagccaa aaacatcctg 1080 gatgttgcac
ggcaattgaa cgatgcccat taa 1113 5 1035 DNA Human tissue 5 atgagagtga
ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt 60
gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc
120 accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa
aagcctgttt 180 accaaggagc ttgaacatgc cctggagaag aatgaagtgg
acctggttgt tcactccttg 240 aaggacctgc ccactgtgct tcctcctggc
ttcaccatcg gagccatctg caagcgggaa 300 aaccctcatg atgctgttgt
ctttcaccca aaatttgttg ggaagaccct agaaaccctg 360 ccagagaaga
gtgtggtggg aaccagctcc ctgcgaagag cagcccagct gcagagaaag 420
ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct tcggaagctg
480 gacgagcagc aggagttcag tgccatcatc ctggcaacag ctggcctgca
gcgcatgggc 540 tggcacaacc gggtggggca gatcctgcac cctgaggaat
gcatgtatgc tgtgggccag 600 ggggccttgg gcgtggaagt gcgagccaag
gaccaggaca tcttggatct ggtgggtgtg 660 ctgcacgatc ccgagactct
gcttcgctgc atcgctgaaa gggccttcct gaggcacctg 720 gaaggaggct
gcagtgtgcc agtagccgtg catacagcta tgaaggatgg gcaactgtac 780
ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac catgcaggct
840 accatccatg tccctgccca gcatgaagat ggccctgagg atgacccaca
gttggtaggc 900 atcactgctc gtaacattcc acgagggccc cagttggctg
cccagaactt gggcatcagc 960 ctggccaact tgttgctgag caaaggagcc
aaaaacatcc tggatgttgc acggcaattg 1020 aacgatgccc attaa 1035 6 1035
DNA Human tissue 6 atgagagtga ttcgcgtggg tacccgcaag agccagcttg
ctcgcataca gacggacagt 60 gtggtggcaa cattgaaagc ctcgtaccct
ggcctgcagt ttgaaatcat tgctatgtcc 120 accacagggg acaagattct
tgatactgca ctctctaaga ttggagagaa aagcctgttt 180 accaaggagc
ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg 240
aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg caagcgggaa
300 aaccctcatg atgctgttgt ctttcaccca aaatttgttg ggaagaccct
agaaaccctg 360 ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag
cagcccagct gcagagaaag 420 ttcccgcatc tggagttcag gagtattcgg
ggaaacctca acacccggct tcggaagctg 480 gacgagcagc aggagttcag
tgccatcatc ctggcaacag ctggcctgca gcgcatgggc 540 tggcacaacc
gggtggggca gatcctgcac cctgaggaat gcatgtatgc tgtgggccag 600
ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct ggtgggtgtg
660 ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa gggccttcct
gaggcacctg 720 gaaggaggct gcagtgtgcc agtagccgtg catacagcta
tgaaggatgg gcaactgtac 780 ctgactggag gagtctggag tctagacggc
tcagatagca tacaagagac catgcaggct 840 accatccatg tccctgccca
gcatgaagat ggccctgagg atgacccaca gttggtaggc 900 atcactgctc
gtaacattcc acgagggccc cagttggctg cccagaactt gggcatcagc 960
ctggccaact tgttgctgag caaaggagcc aaaaacatcc tggatgttgc acggcaattg
1020 aacgatgccc attaa 1035 7 1034 DNA Human tissue 7 atgagagtga
ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt 60
gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc
120 accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa
aagcctgttt 180 accaaggagc ttgaacatgc cctggagaag aatgaagtgg
acctggttgt tcactccttg 240 aaggacctgc ccactgtgct tcctcctggc
ttcaccatcg gagccatctg caagcgggaa 300 aaccctcatg atgctgttgt
cttcacccaa aatttgttgg gaagacccta gaaaccctgc 360 cagagaagag
tgtggtggga accagctccc tgcgaagagc agcccagctg cagagaaagt 420
tcccgcatct ggagttcagg agtattcggg gaaacctcaa cacccggctt cggaagctgg
480 acgagcagca ggagttcagt gccatcatcc tggcaacagc tggcctgcag
cgcatgggct 540 ggcacaaccg ggtggggcag atcctgcacc ctgaggaatg
catgtatgct gtgggccagg 600 gggccttggg cgtggaagtg cgagccaagg
accaggacat cttggatctg gtgggtgtgc 660 tgcacgatcc cgagactctg
cttcgctgca tcgctgaaag ggccttcctg aggcacctgg 720 aaggaggctg
cagtgtgcca gtagccgtgc atacagctat gaaggatggg caactgtacc 780
tgactggagg agtctggagt ctagacggct cagatagcat acaagagacc atgcaggcta
840 ccatccatgt ccctgcccag catgaagatg gccctgagga tgacccacag
ttggtaggca 900 tcactgctcg taacattcca cgagggcccc agttggctgc
ccagaacttg ggcatcagcc 960 tggccaactt gttgctgagc aaaggagcca
aaaacatcct ggatgttgca cggcaattga 1020 acgatgccca ttaa 1034 8 1035
DNA Human tissue 8 atgagagtga ttcgcgtggg tacccgcaag agccagcttg
ctcgcataca gacgggcagt 60 gtggtggcaa cattgaaagc ctcgtaccct
ggcctgcagt ttgaaatcat tgctatgtcc 120 accacagggg acaagattct
tgatactgca ctctctaaga ttggagagaa aagcctgttt 180 accaaggagc
ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg 240
aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg caagcgggaa
300 aaccctcatg atgctgttgt ctttcaccca aaatttgttg ggaagaccct
agaaaccctg 360 ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag
cagcccagct gcagagaagg 420 ttcccgcatc tggagttcag gagtattcgg
ggaaacctca acacccggct tcggaagctg 480 gacgagcagc aggagttcag
tgtcatcatc ctggcaacag ctggcctgca gcgcatgggc 540
tggcacaacc gggttgggca gatcctgcac cctgaggaat gcatgtatgc tgtgggccag
600 ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct
ggtgggtgtg 660 ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa
gggccttcct gaggcacctg 720 gaaggaggct gcagtgtgcc agtagccgtg
catacagcta tgaaggatgg gcaactgtac 780 ctgactggag gagtctggag
tctagacggc tcagatagca tacaagagac catgcaggct 840 accatccatg
tccctgccca gcatgaagat ggccctgagg atgacccaca gttggtaggc 900
atcactgctc gtaacattcc acgagggccc cagttggctg cccagaactt gggcatcagc
960 ctggccaact tgttgctgag caagggagcc aaaaacatcc tggatgttgc
acggcaattg 1020 aacgatgccc attaa 1035 9 1035 DNA Human tissue 9
atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt
60 gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat
tgctatgtcc 120 accacagggg acaagattct tgatactgca ctctctaaga
ttggagagaa aagcctgttt 180 accaaggagc ttgaacatgc cctggagaag
aatgaagtgg acctggttgt tcactccttg 240 aaggacctgc ccactgtgct
tcctcctggc ttcaccatcg gagccatctg caagcgggaa 300 aaccctcatg
atgctgttgt ctttcaccca aaatttgttg ggaagaccct agaaaccctg 360
ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag cagcccagct gcagagaaag
420 ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct
tcggaagctg 480 gacgagcagc aggagttcag tgccatcatc ctggcaacag
ctggcctgca gcgcatgggc 540 tggcacaacc gggtggggca gatcctgcac
cctgaggaat gcatgtatgc tgtgggccag 600 ggggccttgg gcgtggaagt
gcgagccaag gaccaggaca tcttggatct ggtgggtgtg 660 ctgcacgatc
ccgagactct gcttcgctgc atcgctgaaa gggccttcct gaggcacctg 720
gaaggaggtt gcagtgtgcc agtagccgtg catacagcta tgaaggatgg gcaactgtac
780 ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac
catgcaggct 840 accatccatg tccctgccca gcatgaagat ggccctgagg
atgacccaca gttggtaggc 900 atcactgctc gtaacattcc acgagggccc
cagttggctg cccagaactt gggcatcagc 960 ctggccaact tgttgctgag
caaaggagcc aaaaacatcc tggatgttgc acggcaattg 1020 aacgatgccc attaa
1035 10 1034 DNA Human tissue 10 atgagagtga ttcgcgtggg tacccgcaag
agccagcttg ctcgcataca gacggacagt 60 gtggtggcaa cattgaaagc
ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc 120 accacagggg
acaagattct tgatactgca ctctctaaga ttggagagaa aagcctgttt 180
accaaggagc ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg
240 aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg
caagcgggaa 300 aaccctcatg atgctgttgt ctttcaccca aaatttgttg
ggaagaccct agaaaccctg 360 ccagagaaga gtgtggtggg aaccagctcc
ctgcgaagag cagcccagct gcagagaaag 420 ttcccgcatc tggagttcag
gagtattcgg ggaaacctca acacccggct tcggaagctg 480 gacgagcagc
aggagttcag tgccatcatc ctggcaacag ctggcctgca gcgcatgggc 540
tggcacaacc gggtggggca gatcctgcac cctgaggaat gcatgtatgc tgtgggccag
600 ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct
ggtgggtgtg 660 ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa
gggccttcct gaggcacctg 720 gaaggaggct gcagtgtgcc agtagccgtg
catacagcta tgaaggatgg gcaactgtac 780 ctgactggag gagtctggag
tctagacggc tcagatagca tacaagagac catgcaggct 840 accatccatg
tccctgccca gcatgaagat ggccctgagg atgacccaca gttggtaggc 900
atcactgctc gtaacattcc acgagggccc cagttggctg cccagaactt gggcatcagc
960 ctggccaact tgttgctgag caaaggagcc aaaaacatcc tggatgttgc
acggcaatta 1020 acgatgccca ttaa 1034 11 1035 DNA Human tissue 11
atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt
60 gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat
tgctatgtcc 120 accacagggg acaagattct tgatactgca ctctctaaga
ttggagagaa aagcctgttt 180 accaaggagc ttgaacatgc cctggagaag
aatgaagtgg acctggttgt tcactccttg 240 aaggacctgc ccactgtgct
tcctcctggc ttcaccatcg gagccatctg caagcgggaa 300 aaccctcatg
atgctgttgt ctttcaccca aaatttgttg ggaagaccct agaaaccctg 360
ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag cagcccagct gcagagaaag
420 ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct
tcggaagctg 480 gacgagcagc aggagttcag tgccatcatc ctggcaacag
ctggcctgca gcgcatgggc 540 tggcacaacc gggtggggca gatcctgcac
cctgaggaat gcatgtatgc tgtgggccag 600 ggggccttgg gcgtggaagt
gcgagccaag gaccaggaca tcttggatct ggtgggtgtg 660 ctgcacgatc
ccgagactct gcttcgctgc atcgctgaaa gggccttcct gaggcacctg 720
gaaggaggct gcagtgtgcc agtagccgtg catacagcta tgaaggatgg gcaactgtac
780 ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac
catgcaggcc 840 accatccatg tccctaccca gcatgaagat ggccctgagg
atgacccaca gttggtaggc 900 atcactgctc gtaacattcc acgagggccc
cagttggctg cccagaactt gggcatcagc 960 ctggccaact tgttgctgag
caaaggagcc aaaaacatcc tggatgttgc acggcaattg 1020 aacgatgccc attaa
1035 12 3988 DNA Human tissue 12 cacctgacgc gccctgtagc ggcgcattaa
gcgcggcggg tgtggtggtt acgcgcagcg 60 tgaccgctac acttgccagc
gccctagcgc ccgctccttt cgctttcttc ccttcctttc 120 tcgccacgtt
cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 180
gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
240 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
acgttcttta 300 atagtggact cttgttccaa actggaacaa cactcaaccc
tatctcggtc tattcttttg 360 atttataagg gattttgccg atttcggcct
attggttaaa aaatgagctg atttaacaaa 420 aatttaacgc gaattttaac
aaaatattaa cgcttacaat ttccattcgc cattcaggct 480 gcgcaactgt
tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 540
agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
600 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat
tgggtaccgg 660 gccccccctc gaggtcgacg gtatcgataa gcttattaat
gggcatcgtt caattgccgt 720 gcaacatcca ggatgttttt ggctcctttg
ctcagcaaca agttggccag gctgatgccc 780 aagttctggg cagccaactg
gggccctcgt ggaatgttac gagcagtgat gcctaccaac 840 tgtgggtcat
cctcagggcc atcttcatgc tgggcaggga catggatggt agcctgcatg 900
gtctcttgta tgctatctga gccgtctaga ctccagactc ctccagtcag gtacagttgc
960 ccatccttca tagctgtatg cacggctact ggcacactgc agcctccttc
caggtgcctc 1020 aggaaggccc tttcagcgat gcagcgaagc agagtctcgg
gatcgtgcag cacacccacc 1080 agatccaaga tgtcctggtc cttggctcgc
acttccacgc ccaaggcccc ctggcccaca 1140 gcatacatgc attcctcagg
gtgcaggatc tgcccaaccc ggttgtgcca gcccatgcgc 1200 tgcaggccag
ctgttgccag gatgatggca ctgaactcct gctgctcgtc cagcttccga 1260
agccgggtgt tgaggtttcc ccgaatactc ctgaactcca gatgcgggaa ctttctctgc
1320 agctgggctg ctcttcgcag ggagctggtt cccaccacac tcttctctgg
cagggtttct 1380 agggtcttcc caacaaattt tgggtgaaag acaacagcat
catgagggtt ttcccgcttg 1440 cagatggctc cgatggtgaa gccaggagga
agcacagtgg gcaggtcctt caaggagtga 1500 acaaccaggt ccacttcatt
cttctccagg gcatgttcaa gctccttggt aaacaggctt 1560 ttctctccaa
tcttagagag tgcagtatca agaatcttgt cccctgtggt ggacatagca 1620
atgatttcaa actgcaggcc agggtacgag gctttcaatg ttgccaccac actgtccgtc
1680 tgtatgcgag caagctggct cttgcgggta cccacgcgaa tcactctcat
gaattcctgc 1740 agcccggggg atccactagt tctagagcgg ccgccaccgc
ggtggagctc cagcttttgt 1800 tccctttagt gagggttaat ttcgagcttg
gcgtaatcat ggtcatagct gtttcctgtg 1860 tgaaattgtt atccgctcac
aattccacac aacatacgag ccggaagcat aaagtgtaaa 1920 gcctggggtg
cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 1980
ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
2040 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
gcgctcggtc 2100 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
taatacggtt atccacagaa 2160 tcaggggata acgcaggaaa gaacatgtga
gcaaaaggcc agcaaaaggc caggaaccgt 2220 aaaaaggccg cgttgctggc
gtttttccat aggctccgcc cccctgacga gcatcacaaa 2280 aatcgacgct
caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 2340
ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
2400 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
taggtatctc 2460 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
acgaaccccc cgttcagccc 2520 gaccgctgcg ccttatccgg taactatcgt
cttgagtcca acccggtaag acacgactta 2580 tcgccactgg cagcagccac
tggtaacagg attagcagag cgaggtatgt aggcggtgct 2640 acagagttct
tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 2700
tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
2760 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
gcgcagaaaa 2820 aaaggatctc aagaagatcc tttgatcttt tctacggggt
ctgacgctca gtggaacgaa 2880 aactcacgtt aagggatttt ggtcatgaga
ttatcaaaaa ggatcttcac ctagatcctt 2940 ttaaattaaa aatgaagttt
taaatcaatc taaagtatat atgagtaaac ttggtctgac 3000 agttaccaat
gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 3060
atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
3120 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
atcagcaata 3180 aaccagccag ccggaagggc cgagcgcaga agtggtcctg
caactttatc cgcctccatc 3240 cagtctatta attgttgccg ggaagctaga
gtaagtagtt cgccagttaa tagtttgcgc 3300 aacgttgttg ccattgctac
aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 3360 ttcagctccg
gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 3420
gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
3480 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
aagatgcttt 3540 tctgtgactg gtgagtactc aaccaagtca ttctgagaat
agtgtatgcg gcgaccgagt 3600 tgctcttgcc cggcgtcaat acgggataat
accgcgccac atagcagaac tttaaaagtg 3660 ctcatcattg gaaaacgttc
ttcggggcga aaactctcaa ggatcttacc gctgttgaga 3720 tccagttcga
tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 3780
agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
3840 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
catttatcag 3900 ggttattgtc tcatgagcgg atacatattt gaatgtattt
agaaaaataa acaaataggg 3960 gttccgcgca catttccccg aaaagtgc 3988 13
1260 DNA Human tissue 13 cacaggaaac agctatgacc atgattacgc
caagctcgaa attaaccctc actaaaggga 60 acaaaagctg gagctccacc
gcggtggcgg ccgctctaga actagtggat cccccgggct 120 gcaggaattc
atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca 180
gacggacagt gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat
240 tgctatgtcc accacagggg acaagattct tgatactgca ctctctaaga
ttggagagaa 300 aagcctgttt accaaggagc ttgaacatgc cctggagaag
aatgaagtgg acctggttgt 360 tcactccttg aaggacctgc ccactgtgct
tcctcctggc ttcaccatcg gagccatctg 420 caagcgggaa aaccctcatg
atgctgttgt ctttcaccca aaatttgttg ggaagaccct 480 agaaaccctg
ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag cagcccagct 540
gcagagaaag ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct
600 tcggaagctg gacgagcagc aggagttcag tgccatcatc ctggcaacag
ctggcctgca 660 gcgcatgggc tggcacaacc gggttgggca gatcctgcac
cctgaggaat gcatgtatgc 720 tgtgggccag ggggccttgg gcgtggaagt
gcgagccaag gaccaggaca tcttggatct 780 ggtgggtgtg ctgcacgatc
ccgagactct gcttcgctgc atcgctgaaa gggccttcct 840 gaggcacctg
gaaggaggct gcagtgtgcc agtagccgtg catacagcta tgaaggatgg 900
gcaactgtac ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac
960 catgcaggct accatccatg tccctgccca gcatgaagat ggccctgagg
atgacccaca 1020 gttggtaggc atcactgctc gtaacattcc acgagggccc
cagttggctg cccagaactt 1080 gggcatcagc ctggccaact tgttgctgag
caaaggagcc aaaaacatcc tggatgttgc 1140 acggcaattg aacgatgccc
attaataagc ttatcgatac cgtcgacctc gagggggggc 1200 ccggtaccca
attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa 1260 14 32
DNA Artificial Sequence Description of Artificial Sequence Primer
for PCR amplification 14 cgtggaattc atgagagtga ttcgcgtggg ta 32 15
47 DNA Artificial Sequence Description of Artificial Sequence
Primer for PCR amplification 15 ggagaagctt attaatgggc atcgttcaat
tgccgtgcaa catccag 47 16 20 DNA Artificial Sequence Description of
Artificial Sequence Primer for PCR amplification 16 tcgcctccct
ctagtctctg 20 17 21 DNA Artificial Sequence Description of
Artificial Sequence Primer for PCR amplification 17 cagcaggagt
tcagtgccat c 21 18 21 DNA Artificial Sequence Description of
Artificial Sequence Primer for PCR amplification 18 gatggcactg
aactcctgct g 21 19 20 DNA Artificial Sequence Description of
Artificial Sequence Primer for PCR amplification 19 cagcaaccca
ggcatctgtg 20 20 22 DNA Artificial Sequence Description of
Artificial Sequence Primer for PCR amplification 20 gtaatacgac
tcactatagg gc 22 21 22 DNA Artificial Sequence Description of
Artificial Sequence Primer for PCR amplification 21 ctaaagggaa
caaaagctgg ag 22 22 21 DNA Artificial Sequence Description of
Artificial Sequence Primer for PCR amplification 22 cagctatgac
catgattacg c 21 23 54 DNA Artificial Sequence CDS (32)..(52)
Description of Artificial Sequence Modified 5' untranslated region
of plasmid pExp1 (P 27, L 17-25) 23 aattctaaca taagttaagg
aggaaaaaaa a atg aga gtt att cgt gtc ggt ac 54 Met Arg Val Ile Arg
Val Gly 1 5 24 7 PRT Artificial Sequence Description of Artificial
Sequence N-terminal of peptide encoded by ID 23; related to
N-terminal of PBGD (P 27, L 23-27) 24 Met Arg Val Ile Arg Val Gly 5
25 54 DNA Artificial Sequence Description of Artificial Sequence
Primer for PCR amplification 25 aattctaaca taagttaagg aggaaaaaaa
aatgagagtt attcgtgtcg gtac 54 26 46 DNA Artificial Sequence
Description of Artificial Sequence Primer for PCR amplification 26
cgacacgaat aactctcatt tttttttcct ccttaactta tgttag 46 27 32 DNA
Artificial Sequence Description of Artificial Sequence Primer for
PCR amplification 27 gatcactcat gtttgacagc ttatcatcga tt 32 28 30
DNA Artificial Sequence Description of Artificial Sequence Primer
for PCR amplification 28 agctaatcga tgataagcgt caaacatgag 30 29 32
DNA Artificial Sequence Description of Artificial Sequence Primer
for PCR amplification 29 agtcagaatt cagacgcacg gcggtacgat aa 32 30
32 DNA Artificial Sequence Description of Artificial Sequence
Primer for PCR amplification 30 attcactcga ggtcaccatc ggtaccagtt ca
32 31 32 DNA Artificial Sequence Description of Artificial Sequence
Primer for PCR amplification 31 agatcaagct tcggccagac gcaggttatc ta
32 32 34 DNA Artificial Sequence Description of Artificial Sequence
Primer for PCR amplification 32 atacactcga gaccggcatg agtatccttg
tcac 34 33 30 DNA Artificial Sequence Description of Artificial
Sequence Primer for PCR amplification 33 actgacctcg agcggcacgt
aagaggttcc 30 34 29 DNA Artificial Sequence Description of
Artificial Sequence Primer for PCR amplification 34 actgaactcg
agaattacgc cccgccctg 29 35 60 DNA Artificial Sequence CDS (1)..(60)
Description of Artificial Sequence Primer for PCR amplification 35
atg tct ggt aac ggc att gcg gct gca acg gcg gaa gaa aac agc cca 48
Met Ser Gly Asn Gly Ile Ala Ala Ala Thr Ala Glu Glu Asn Ser Pro 1 5
10 15 aag atg aga gtg 60 Lys Met Arg Val 20 36 20 PRT Artificial
Sequence Description of Artificial Sequence Primer for PCR
amplification 36 Met Ser Gly Asn Gly Ile Ala Ala Ala Thr Ala Glu
Glu Asn Ser Pro 1 5 10 15 Lys Met Arg Val 20 37 25 DNA Artificial
Sequence Description of Artificial Sequence Fragment of normal
chromosomal sequence 37 agcgcatggg ctggcacaac cgggt 25 38 9 PRT
Artificial Sequence Description of Artificial Sequence encoded by
SEQ ID NO37 38 Gln Arg Met Gly Trp His Asn Arg Val 1 5 39 25 DNA
Artificial Sequence Description of Artificial Sequence Fragment of
AIP chromosomal sequence 39 agcgcatggg ctagcacaac cgggt 25 40 68
DNA Artificial Sequence Description of Artificial Sequence linear
single-stranded chimeric (RNA/DNA) oligonucleotide with hairpin
secondary structure 40 agcgcatggg ctggcacaac cgggttttta cccggttgtg
ccagcccatg cgctccgggt 60 tttcccgg 68
* * * * *