Note: Descriptions are shown in the official language in which they were submitted.
1 338643
PURIFICATION AND ADMINISTRATION OF DNA REPAIR ENZYMES
BACKGROUND OF THE lNV~NLlON
1. Field of the Invention
This invention relates to DNA repair enzymes and, in
particular, to 1) methods for purifying DNA repair enzymes, and 2)
methods and means for administering DNA repair enzymes to living
cells in situ, e.g. human skin cells, so that the enzymes can
enter the cells and enhance the repair of damaged DNA in the
cells.
2. Description of the Prior Art
Skin cancer is a serious human health problem. The incidence
of non-melanoma skin cancer in the United States is 500,000 per
year, and 23,000 per year for melanoma. Annual deaths are 2,000
and 6,000 respectively, and 800,000 deaths from skin cancer are
predicted in the next 88 years if current trends continue.
The causal link between non-melanoma skin cancer and
ultraviolet light exposure from the sun has been clearly
established, and sun exposure is an important causative factor in
melanoma. The target for ultraviolet light damage leading to
cancer is widely accepted as DNA.
Xeroderma pigmentosum is a human genetic disease in which
patients develop solar damage, pigmentation abnormalities and
malignancies in sun-exposed skin. A review of the disease was
authored by J.H. Robbins, K.H. Kraemer, M.A. Lutzner, B.W. Festoff
and H.G. Coon, entitled "Xeroderma Pigmentosum: An Inherited
Disease with Sun Sensitivity, Multiple Cutaneous Neoplasms, and
Abnormal DNA Repair", and published in the ANNALS OF INTERNAL
MEDICINE, volume 80, number 2, pages 221-248, February, 1974. The
disease occurs in 1 of 250,000 worldwide. Cells from xeroderma
pigmentosum patients are deficient in repair of ultraviolet damage
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to DNA, which results in a cancer incidence 4,800 times the
frequency of the general U.S. population. There is no cure, and
treatment consists of avoiding sun exposure and excising skin
lesions. Death occurs 30 years earlier in these patients than
among the general U.S. population.
Research into the basic mechanisms of DNA repair has
established outlines of biochemical pathways which remove
ultraviolet damage in DNA. Bacterial repair systems have been
demonstrated to differ significantly from repair in human cells.
However, the enzyme endonuclease V (also referred to herein as T4
endonuclease V and denV endonuclease V) has the ability to enhance
DNA repair in human cells as evidenced by increased UV-specific
incision of cellular DNA, increased DNA repair replication, and
increased UV survival after treatment with the enzyme.
The endonuclease V enzyme is produced by the denV gene of the
bacteriophage T4. It has been established that this enzyme
catalyzes the rate limiting, first step in the removal of
UV-induced DNA damage, namely, single strand incision of DNA at
the site of damage. In particular, the enzyme exhibits
glycosylase and apurinic/apyrimidinic endonuclease activities and
acts at the site of ultraviolet induced pyrimidine dimers. See
"Evidence that the UV Endonuclease Activity Induced by
Bacteriophage T4 Contains Both Pyrimidine Dimer-DNA Glycosylase
and Apyrimidinic/Apurinic Endonuclease Activities in the Enzyme
Molecule" by H.R. Warner, L.M. Christensen and M.L. Persson, in
JOURNAL OF VIROLOGY, 1981, Vol. 40, pages 204-210; "denV Gene of
Bacteriophage T4 Codes for Both Pyrimidine Dimer DNA Glycosylase
and Apyrimidinic Endonuclease Activities" by S. McMillan, H.J.
Edenberg, E.H. Radany, R.C. Friedberg and E.C. Friedberg, in
JOURNAL OF VIROLOGY, 1981, Vol. 40, pages 211-223, and "Physical
Association of Pyrimidine Dimer DNA Glycosylase and
Apurinic/Apyrimidinic DNA Endonuclease Essential for Repair of
Ultraviolet-damaged DNA" by Y. Nakabeppu and M. Sekiguchi, in
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, 1981, Vol. 78,
pages 2742-2746.
~3~ 1 3 3 8 6 4 3
Other enzymes having the ability to repair DNA damage have
also been identified. These enzymes include O6-methylguanine-DNA
methyltransferases, photolyases, uracil- and hypoxanthine-DNA
glycosylases, apyrimidinic/apurinic endonucleases, DNA
exonucleases, damaged-bases glycosylases (e.g.,
3-methyladenine-DNA glycosylase), correndonucleases alone or in
complexes (e.g., E. coli uvrA/uvrB/uvrC endonuclease complex), and
other enzymes and enzyme complexes whose activities at present are
only partially understood, such as, the products of the ERCC genes
of humans and the RAD genes of yeast. Various of these enzymes
have been purified to homogeneity from microorganisms, and the
genes for some of the enzymes have been cloned. As used herein,
the term "DNA repair enzymes" is intended to include the foregoing
enzymes, the T4 endonuclease V enzyme, and other enzymes now known
or subsequently discovered or developed which have the ability to
participate in repair of damaged nucleic acids and, in particular,
damaged DNA.
To date, the use of exogenous enzymes in DNA repair systems
has been limited to laboratory experiments designed to study the
biochemical and evolutionary relationships among DNA repair
pathways. Clinical application of these laboratory results has
not been undertaken because, inter alia, there has been no
effective way of purifying commercial quantities of DNA repair
enzymes, and there has been no effective, non-toxic way of
~' ;n;ctering DNA repair enzymes to living cells. The present
invention addresses both of these long-standing problems in the
art.
Purification of DNA enzymes for commercial use requires a
homogenous final product, high yield, speed, simplicity and low
cost. The existing methods of the art have been unable to meet
these goals, as follows:
(1) P. Seawell, E.C. Friedberg, A.K. Ganesan and P.C.
Hanawalt, "Purification of Endonuclease V of Bacteriophage T4" in
DNA REPAIR: A LABORATORY MANUAL OF RESEARCH PROCEDURES, edited by
~4~ 1 3 3 8 6 4 3
E.C. Friedberg and P.C. Hanawalt, Marcel Dekker, Inc., New York,
1981, Volume 1, Part A, pages 229-236.
This method uses phage T4 infected E. coli, and purification
relies on phase-separation and two ion-exchange chromatography
steps (DEAE- and phospho-cellulose). The DEAE chromatography step
limits the yield of the method because all proteins must bind in
order to elute the enzyme of interest. The method is not rapid:
each chromatography step is preceded by dialysis, each elution
requires at least 20 hours, and each fraction is assayed for
activity. The process is neither simple nor inexpensive: tedious
phase separation and repetitive assays are performed, and all
spent dialysate and separated phases are discarded.
Significantly, the authors of this method describe their final
product as being only partially purified.
The basic steps of the Seawell et al. method were first
described by E.C. Friedberg and J.J. King in "Dark Repair of
Ultraviolet-irradiated Deoxyribonucleic acid by Bacteriophage T4:
Purification and Characterization of a Dimer-Specific
Phage-Induced Endonuclease", JOURNAL OF BACTERIOLOGY, 1971, Vol.
106, pages 500-507. This earlier version of the method included
an additional DNA-cellulose step, which was omitted in the later
version. A method similar to the Friedberg and King method was
described by S. Yasuda and M. Sekiguchi, "T4 Endonuclease Involved
in Repair of DNA" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES,
December, 1970, Vol. 67, pages 1839-1845. Instead of using a
DNA-cellulose step as in the Friedberg and King method, the Yasuda
and Sekiguchi method included an optional gel filtration step.
(2) Y. Nakabeppu, K. Yamashita and M. Sekiguchi,
"Purification and Characterization of Normal and Mutant Forms of
T4 Endonuclease V" JOURNAL OF BIOLOGICAL CHEMISTRY, 1982, Vol.
257, pages 2556-2562.
The basic steps of this method were first described by S.
Yasuda and M. Sekiguchi, "Further Purification and
Characterization of T4 Endonuclease V", BIOCHIMICIA ET BIOPHYSICA
ACTA, 1976, Vol. 442, pages 197-207. These methods are similar to
-
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1 338643
the Seawell et al. method, except that they substitute cation
exchange (carboxymethyl Sephadex (Trademark)) chromatography for
anion exchange (DEAE) chromatography, and add additional
chromatography steps including either hydroxylapatite or gel
filtration and UV DNA cellulose (the Yasuda and Sekiguchi method
also differs from the Seawell et al. method in that it does not
include a phosphocellulose step). These methods have the same
difficulties as the Seawell et al. method, with the additional
problems of lower yield, less speed and simplicity, and greater
cost.
(3) K.M. Higgins and R.S. Lloyd, "Purification of the T4
Endonuclease V", MUTATION RESEARCH, 1987, Vol. 183, pages 117-121.
This method uses an E. coli strain which harbors a plasmid
containing the phage T4 denV structural gene under the control of
the phage lambda rightward promoter. The chromatography steps are
single-stranded DNA agarose, chromatofocusing and cation exchange
(carboxymethyl-Sephadex (Trademark)). The yield is low compared
to the present invention, in that 12 liters of bacteria are
required for 15 mg pure enzyme. The yield is also limited by the
requirement that all proteins bind to the chromatofocusing column
in order to elute the desired enzyme. The method is not rapid:
each chromatography step is preceded by dialysis and concentration
by ultrafiltration; at least two of the steps require on the order
of 17.5 hours for elution; and each step is followed both by
enzyme activity assays and polyacrylamide gel analysis of each
fraction. The method is not simple: the single-stranded DNA
agarose chromatography requires pooling of 84% of the collected
fraction (520 ml of 700 ml eluent), extensively diluting the
loaded protein; experiments in connection with the present
invention showed that the chromatofocusing step was not
reproducible using DEAE agarose and Servalyte (Trademark)
ampholines; ultrafiltration is required in addition to dialysis;
and tedious, repetitive activity assays and gel analysis are
performed after each step. The method is expensive: large
ultrafiltration devices are used and discarded at every step; the
single-stranded DNA agarose is exposed to crude bacterial lysates
with active nucleases which drastically reduce
.: ' ~
-6- l 3 3 8 6 4 3
the useful life of the column; and costly chromatofocusing
reagents including Pharmacia (Trademark) PBE 94 gel and polybuffer
ampholines must be used.
In addition to the foregoing, two methods have been published
for the purification of O -methylguanine-DNA methyltransferase.
See B. Demple, A. Jacobsson, M. Olsson, P. Robbins and T. Lindahl,
"Repair of Alkylated DNA in Escherichia coli: Physical properties
of O -methylguanine-DNA methyltransferase" in THE JOURNAL OF
BIOLOGICAL CHEMISTRY, vol. 257, pages 13776-13780, 1982, and Y.
Nakabeppu, H. Kondo, S. Kawabata, S. Iwanaga and M. Sekiguchi,
"Purification and Structure of the Intact Ada Regulatory Protein
of Escherichia coli K12 0 -Methylguanine-DNA Methyltransferase" in
THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 260, pages 7281-7288,
1986. The Demple method uses phosphocellulose chromatography
before DNA-cellulose and gel filtration, and includes a final
phenylagarose chromatography step. The Nakabeppu method uses two
rounds of ion-exchange (DEAE-) chromatography followed by
phosphocellulose and gel filtration chromatography.
A general review of purification methods for DNA repair
enzymes can be found in DNA REPAIR: A LABORATORY MANUAL OF
RESEARCH PROCEDURES, edited by E. Friedberg and P. C. Hanawalt,
published by Marcel Dekker, New York. Volume I, part A, of this
text contains methods for purifying five enzymes: photolyase,
endonuclease V (discussed above), AP endonuclease, uracil-DNA
glycosylase and hypoxanthine-DNA glycosylase, in chapters 18-22,
respectively. Volume II, chapters 3-5, discuss the Demple method
referred to above and methods for purifying 3-methyladenine-DNA
glycosylases. Volume III, Section IV, contains methods for
purification of photolyase, the uvrABC excision nuclease and the
uvrD helicase in chapters 23-25. None of these methods, nor the
two methods discussed above for purifying 06-methylguanine-DNA
methyltransferase, use the purification procedures of the present
invention.
Various approaches have been considered in the field of DNA
repair for delivering DNA repair enzymes to ~ n cells. The
_7_ 1 3 3 8 6 4 3
goal of these efforts has been to discover and characterize the
pathways of DNA repair in mammalian cells and their evolution, not
to develop commercial methods for augmenting DNA repair. Thus,
researchers have not used normal cells, such as skin epidermal
keratinocyte cells, as target cells, but rather have concentrated
on fibroblasts from patients with xeroderma pigmentosum.
Similarly, prior research has focused on non-physiological
techniques for introducing DNA repair enzymes into cells which are
useful only in the laboratory and which compromise the physiology
of the target cells. The published reports regarding this work
include:
(1) K. Tanaka, M. Sekiguchi and Y. Okada, "Restoration of
ultraviolet-induced unscheduled DNA synthesis of xeroderma
pigmentosum cells by the concomitant treatment with bacteriophage
T4 endonuclease V and HVJ (Sendai virus)", PROCEEDINGS OF THE
NATIONAL ACADEMY OF SCIENCES U.S.A., 1975, Vol. 72, pages
4071-4075; and K. Tanaka, H. Hayakawa, M. Sekiguchi and Y. Okada,
"Specific action of T4 endonuclease V on damaged DNA in xeroderma
pigmentosum cells in vivo", PROCEEDINGS OF THE NATIONAL ACADEMY OF
SCIENCES U.S.A., 1977, Vol. 74, pages 2958-2962.
In these two reports, fibroblasts derived from patients with
xeroderma pigmentosum were treated with inactivated Sendai virus
and endonuclease V after W irradiation. Proteins on the coat of
the Sendai virus rendered the cells permeable to endonuclease V.
This treatment enhanced DNA repair replication and increased
survival of the treated cells. This method of introducing the
enzyme is not practical for commercial application because of the
pathogenicity of the Sendai virus. Large external enzyme
concentrations are also required. In its discussion section, the
Tanaka et al. reference discusses approaches to the study of the
evolution of macromolecular (i.e. DNA repair) systems in organisms
and mentions liposome methods and erythrocyte ghost/HVJ methods as
other methods for introducing macromolecules into cells.
Significantly, Tanaka et al. ultimately conclude that the Sendai
virus method is the most simple and applicable method in basic
-8- 1 3 3 8 6 4 3
research for the introduction of rather small macromolecules of
about 20,000 daltons, i.e., the T4 endonuclease V molecule.
(2) G. Ciarrocchi and S. Linn, "A cell-free assay measuring
repair DNA synthesis in human fibroblasts", PROCEEDINGS OF THE
NATIONAL ACADEMY OF SCIENCES U.S.A., 1978, Vol. 75, pages
1887-1891.
In this report, human normal and xeroderma pigmentosum
fibroblasts were disrupted by osmotic shock after UV irradiation,
and incubated with endonuclease V. DNA repair synthesis was
increased in both types of cells, and repair in xeroderma
pigmentosum cells increased to the level of normal cells. This
method for introducing enzyme into cells was only employed for in
vitro research, as it destroys the integrity of the cell membrane
and viability is drastically affected. Large external enzyme
concentrations are also required.
(3) D. Yarosh and R. Setlow, "Permeabilization of
Ultraviolet-irradiated Chinese hamster cells with polyethylene
glycol and introduction of ultraviolet endonuclease from
Micrococcus luteus", MOLECULAR AND CELLULAR BIOLOGY, 1981, Volume
1, pages 237-244.
In this method, hamster cells were treated with polyethylene
glycol after UV irradiation and then incubated with a DNA repair
enzyme which acts similarly to endonuclease V. The enzyme entered
the cells and acted on resident DNA. The method was toxic to
target cells, probably because it relied on permeabilization, and
vital molecules exited as the enzyme entered. This method also
requires large external enzyme concentrations for efficacy.
(4) J.H.J. Hoeijmakers, "Characterization of genes and
proteins involved in excision repair of human cells", JOURNAL OF
CELL SCIENCE SUPPL., 1987, Vol. 6, pages 111-125.
This reference summarizes a body of research in which
proteins were introduced into the nuclei of cells by
microinjection. When endonuclease V was injected into the nuclei
of xeroderma pigmentosum cells, DNA repair synthesis was
-9- 1 3 3 8 6 4 3
increased. This method is applicable only for laboratory
research.
(5) K. Valerie, A.P. Green, J.K. de Riel and E.E. Henderson,
"Transient and stable complementation of ultraviolet repair in
xeroderma pigmentosum cells by the denV gene of bacteriophage T4",
CANCER RESEARCH, 1987, Vol. 47, pages 2967-2971.
In this method, the denV gene under the control of a
r~ ~;an promoter was transfected into xeroderma pigmentosum
cells. Clones selected for uptake of the denV gene showed
increased incision of UV-DNA, enhanced DNA repair synthesis and
increased resistance to ultraviolet irradiation. The transfection
process is very inefficient (less than one success per million
cells) for normal human cells. These methods fall into the
category of gene therapy, and are beyond the scope of the current
art for commercial use.
In a few laboratories, liposomes have been used in the
topical delivery of drugs, but not of enzymes and, in particular,
not of DNA repair enzymes. The reports of encapsulation and
topical drug delivery include:
Delivery of triaminolone: Michael Mezei and Vijeyalakshmi
Gulasekharam, "Liposomes - A selective drug delivery system for
the topical route of administration, I. lotion dosage form", in
LIFE SCIENCES, volume 26, pages 1473-1477, 1980; Michael Mezei and
Vijeyalakshmi Gulasekharam, "Liposomes - A selective drug delivery
system for the topical route of administration: gel dosage form",
in JOURNAL OF PHARMACY AND PHARMACOLOGY, volume 34, pages 473-474,
1981. Delivery of tetracaine: Adrienn Gesztes and Michael Mezei,
"Topical anaesthesia of the skin by liposome-encapsulated
tetracaine", ANESTHESIA AND ANALGESIA, volume 67, pages 1079-1081,
1988. Delivery of methotrexate: H.M. Patel "Liposomes as a
controlled-release system", in BIOCHEMICAL SOCIETY TRANSACTIONS,
volume 13, pages 513-516, 1985. Delivery of hydrocortisone: W.
Wohlrab and J. Lasch, "Penetration kinetics of liposomal
hydrocortisone in human skin", in DERMATOLOGICA, volume 174, pages
18-22, 1987.
1 338643
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The use of pH sensitive liposomes to mediate the cytoplasmic
delivery of calcein and FITC dextran has been described in the
following references: Robert Straubinger, Keelung Hong, Daniel
Friend and Demetrios Papahadjopoulos, "Endocytosis of Liposomes
and Intracellular Fate of Encapsulated Molecules: Encounter with a
Low pH Compartment after Internalization in Coated Vesicles,"
CELL, volume 32, pages 1069-1079, 1983; and Robert Straubinger,
Nejat Duzgunes and Demetrios Papahadjopoulos, "pH-Sensitive
Liposomes Mediate Cytoplasmic Delivery of Encapsulated
Macromolecules," FEBS LETTERS, volume 179, pages 148 - 154, 1985.
Other discussions of pH sensitive liposomes can be found in
chapter 11 of the book CELL FUSION, edited by A.E. Sowers,
entitled "Fusion of Phospholipid Vesicles Induced by Divalent
Cations and Protons" by Nejat Duzgunes, Keelung Hong, Patricia
Baldwin, Joe Bentz, Shlomo Nir and Demetrios Papahadjopoulos,
published by Plenum Press, N.Y., 1987, pages 241 - 267. See also
Ellens, Bentz and Szoka, "pH-Induced destabilization of
phosphatidylethanolamine-containing liposomes: role of bilayer
contact," BIOCHEMISTRY, volume 23, pages 1532-1538, 1984, and
Bentz, Ellens and Szoka, "Destabilization of
Phosphatidylethanolamine-Cont~;n;ng Liposomes: Hexagonal Phase and
Asymmetric Membranes", BIOCHEMISTRY, volume 26, pages 2105-2116,
1987. None of these references discusses or suggests the use of
pH sensitive liposomes to topically administer DNA repair enzymes
to human skin.
SUMMARY OF THE lNV~NLlON
In accordance with certain of its aspects, the invention
provides a process for purifying a DNA repair enzyme comprising:
(a) contacting an aqueous solution of the DNA repair enzyme
in an impure state with a molecular sieve having an exclusion
limit such that the DNA repair enzyme will pass through the
molecular sieve at a slower rate than at least some of the
impurities in the aqueous solution;
(b) isocratically eluting the DNA repair enzyme from the
molecular sieve with an aqueous elution solution so as to obtain
~ -11- 1 338643
the DNA repair enzyme in one or more ~Plec~e~ fractions of the
eluate in a state of ~ ~1 purity, said ~ elution
solution being chosen so that complexes can form he~e~n the DN~
repair enzyme and selected nucleic acids:
(c) without further purification contacting the one or more
selected fractions of step (b) in said ~ elution solution
with one or more selected ~l~le;~ acids which have been immobilized
on a solid support so as to form imm~h;l; 7~ e; ~-acid/DN~-
repair-enzyme complexes hP~2^~ the DN~ repair enzyme and the one
or more selected, immobilized ~llrle;~ acids;
(d) washing the immobilized complexes to remove at least
some of the remaining impurities; and
(e) eluting the DNA repair enzyme fram the one or more
selected, immobilized nucleic acids so as to obtain the DN~ repair
en2yme in one or more selected fractions of the eluate in a state
of further erbanced purity.
More part; ~11 ~rly, the steps of the purification process
comprise:
(a) applying an ~ yl~ solution of the DN~ repair enzyme
in an impure state (e.g., an extract of cells which have been
genetically engineered to produce the DN~ repair enzyme) to a
molecular sieve (e.g., a gel filtration column) having a mean pore
size such that the exclusion limit (nE#~mcd either by molecular
weight or by Stokes' radius) is:
(i) larger than the nE#~xncd r~leoll~r weight or Stokes'
radius of the DNA repair enzyme (e.g., in the case of
T4 endonuclease V, an exclusion limit larger than about
16,500 daltons or about 18 Angstroms), and
(ii) smaller than the estimated molecular weight or Stokes'
radius of at least some of the impurities (e.g., an
exclusion limit smaller than about 60,000 daltons or
about 35 A~LLul.~);
(b) isocratically eluting the DN~ repair enzyme from the
molecular sieve with an elution buffer so as to obtain the DN~
repair enzyme in one or more selected fractions of the eluate in a
state of enhanced purity, the elution buffer being chosen so that
1 338643
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complexes can form between the DNA repair enzyme and selected
nucleic acids;
(c) contacting the one or more selected fractions of step
(b) with one or more selected nucleic acids (e.g., single-stranded
DNA) which have been immobilized on a solid support (e.g.,
CNBr-activated Sepharose (Trademark)) so as to form immobilized
nucleic-acid/DNA-repair-enzyme complexes between the DNA repair
enzyme and the one or more selected, immobilized nucleic acids;
(d) washing the immobilized complexes (e.g., with the
elution buffer) to remove at least some of the remaining
impurities; and
(e) eluting the DNA repair enzyme from the one or more
selected, immobilized nucleic acids with an elution buffer
containing a gradient of a material (e.g., NaCl) capable of
disassociating the DNA repair enzyme from the one or more
selected, immobilized nucleic acids so as to obtain the DNA repair
enzyme in one or more selected fractions of the eluate in a state
of further enhanced purity.
As demonstrated by the examples presented below, in the
preferred embodiments of the invention, the DNA repair enzyme is
obtained as a homogeneous protein at the end of step (e).
This purification process takes advantage of two highly
specific characteristics of most DNA repair enzymes: their small
size and their affinity for nucleic acids, in particular,
single-stranded DNA. Proteins are separated by molecular sieve
(gel) filtration, excluding the vast majority of proteins larger
than DNA repair enzymes, while retarding the elution of DNA repair
enzymes. Because the filtration column is eluted isocratically
with almost any buffer, the retarded proteins can be loaded
directly onto a nucleic acid affinity column without assay,
dialysis or concentration. The nucleic acid affinity column is
then developed with either non-specific changes (e.g., gradients
of salt, pH, detergent, voltage or temperature) or specific
changes (e.g., competing ligand), eluting the DNA repair enzyme in
a concentrated form. Since the condition required to elute a
,r,i~
1 338643
-13-
protein from a nucleic acid affinity column is a unique
characteristic of the protein, the peak of pure DNA repair enzyme
can be pooled without assay. The process thus meets the goals of
commercial protein purification: it produces homogenous, pure
enzyme; it has high yield because only the desired proteins are
retained on the chromatography columns; it is rapid and can be
completed in one day; it is simple and requires no dialysis or
activity assays; and it is inexpensive in not consuming disposable
reagents in dialysis or assays and in protecting nucleic acid
affinity columns from crude cell lysates.
As fully described in the examples set forth below, the
purification procedure of the invention has been successfully
applied to the purification of the T4 endonuclease V enzyme. In
outline, E. coli harboring a plasmid with the denV structural gene
under the control of the TAC promoter were grown to log phase and
denV gene expression was induced by the addition of
isopropylthiogalactopyranoside. A cell lysate was prepared,
concentrated and dialyzed against the buffer subsequently used in
both gel filtration and DNA affinity chromatography. The pore
diameter of the gel filtration media was selected to exclude from
the gel most contaminating proteins while including the desired T4
endonuclease V protein. The cell lysate was applied to the gel
filtration column and eluted isocratically, the excluded proteins
being discarded and the retained proteins being collected. The
retained proteins were then applied directly to a single-stranded
DNA agarose column. The column was washed and then developed with
a salt gradient. The eluent was monitored for optical density at
280 nm and the peak of optical density was pooled. This peak
comprised the desired denV endonuclease V enzyme which was
subsequently shown to have been purified to homogeneity.
In addition to the foregoing purification procedures, the
present invention also provides methods and means for
administering DNA repair enzymes to living cells. In particular,
in accordance with these aspects of the invention, DNA repair
enzymes are encapsulated in liposomes to form pharmaceutical
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preparations suitable for administration to living cells and, in
particular, suitable for topical ~' ;n~stration to human skin.
When delivered to human cells in this form, the DNA repair enzymes
will enter the cells, incise damaged DNA, enhance DNA repair
synthesis and increase cell survival after exposure to ultraviolet
light.
In comparison with prior art methods, the delivery system of
the invention has the advantages of requiring high enzyme
concentration only within the liposomes and not in the general
exterior of the cells, and of delivering the enzyme while
preserving the integrity of the target cells. Also, by suitable
modifications of the liposome membranes, the liposomes can be made
to bind to specific sub-populations of cells, thereby increasing
the efficiency and/or specificity of enzyme delivery. As result
of these improvements, the invention allows DNA repair enzymes to
be used clinically, either before or after exposure to ultraviolet
light, to help combat skin cancer caused by UV-damaged DNA in both
normal individuals and patients suffering from xeroderma
pigmentosum.
As fully described in the examples presented below, the
delivery system of the invention has been successfully used to
administer endonuclease V to normal human epidermal keratinocytes
and fibroblasts, transformed human normal and xeroderma
pigmentosum cells, and to living skin. In outline, a lipid
mixture was dissolved in organic solvents and dried to a thin film
in a glass vessel. In certain preferred embodiments, the lipid
mixture was chosen to produce liposomes which were pH sensitive.
Endonuclease V which had been purified in accordance with the
methods of the present invention was added to the vessel at high
concentration in an aqueous buffer to rehydrate the lipid. The
mixture was then agitated by vortexing and sonicated to form
liposomes. The spheres were then separated from unincorporated
enzyme by centrifugation or gel filtration. The liposomes were
then diluted into media and added to the target cells.
Alternatively, in the case of living skin, the liposomes were
-15- 1 3 3 8 6 4 3
suspended in a lotion, and the lotion was ~1;P~ to the skin.
m e addition of the liposomes to the ~lls resulted in ~ 31
DNA repair as ev;~ r~ by i ~-~ase~ W -~per-;f;c ;nr.;~;nn,
increased DN~ repair replication, and incre3sed W -sNrvival.
In a~o~L~k~e with these ~p ctc, the invention provides a
process for pro~l~;n~ a pharm~Pllt;~l preparation of a DNA
repair enzyme comprising:
(a) grcwing cells which synth~c;7.~ the DNA repair enzyme;
(b) preparing an extract of the cells which contains the
DNA repair enzyme in an impure state;
(c) contacting the extract with a mnl e~ r sieve having
an exclusion limit such that the DN~ repair enzyme will pass
through the molecular sieve at a slower rate than at least some
of the impurities in the extract;
(d) isocratically eluting the DNA repair enzyme from the
molecular sieve with an ~lP~l~ n ~olllt;~n so as to obtain
the DNA repair enzyme in one or more ~Plecte~ fractions of the
eluate in a state of enhanoel purity, said ~ 1 llt;nn
solution being chosen so that complexes can form he~7~^n the DNA
repair enzyme and selected nucleic acids;
(e) without further purification, contacting the one or
more selected fractions fram step (d) in said ~l~al~ elution
solution with one or more selected nucleic acids which have been
immobilized on a solid support so as to form immob;l;7.e~ nucleic-
acid/DNA-repair-enzyme complexes hp-~7~cn the DN~ repair enzyme
and the one or more selected, imm~h;l;7e~ acids;
(f) washing the imm~h;l; 7e~ complexes to remove at least
some of the remaining impurities;
(g) eluting the DNA repair enzyme from the one or more
selected, immobilized ~1~.1 e;c acids so as to obtain the DNA
repair enzyme in one or more selected fractions of the eluate in
a state of further enhanoed purity; and
(h) e ~ ting at least a portion of the purified DN~
repair enzyme contained in the one or more selected fractions of
step (g) in li~ s and combining the liposomes with a vehicle
to form the ph~rm~r~-lltical preparation.
E31.~
-15a- ~ 338643
In a~ ku~ with ~ ~ U~ A~pect of the invention, there
is provided a methcd for adr~lIJsbering a pratein having
intracellular binlog;~l activity into the interior of living
skin cells which lie below the skin's stratum corneum, ccmprising
the steps of:
(a) ~ ting the protein in liposomes; and
(b) applying the liposomes to the outer surface of living
skin so that the protein ~ ted in the liposomes traverses
the skin's stratum cnrnPI~ and the outer mem~L~ ~ of said cells
and is thereby delivered by the l;lf~ ~ into the interior of
said cells.
Also according to the invention, there is provided a
ph~rm~c~lltical preparation for ~ tion to the outer surface
of living skin comprising liposomes having a protein having
intracellular biological activity ~ ted therein.
Further according to the invention, there is provided a
method of preparing a protein for ~ tion to the outer
surface of living skin comprising the step of ~ ting the
protein in ~ Y~ ~S.
- 1 338643
-16-
The invention also provides a pharmaceutical preparation
comprising liposomes having a DNA repair enzyme encapsulated
therein.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the results of polyacrylamide gel
electrophoresis of T4 endonuclease V and O -methylguanine-DNA
methyltransferase purified by the present invention. Twenty-five
ug of purified T4 endonuclease V (left lane) and 25 ug of purified
methyltransferase (center lane) were denatured with SDS, loaded on
a 15% discontinuous polyacrylamide gel and electrophoresed with
molecular weight markers (right lane). The size of the molecular
weight markers from the top were: 67,000; 45,000; 36,000; 29,000;
24,000; 20,100; and 14,200. The proteins in the gel were stained
with Coomassie (Trademark) blue. The gel shows that each protein
was homogenous and pure and of the appropriate size (16,500 for T4
endonuclease V and 19,000 for methyltransferase).
Figure 2 shows the enzyme activity of T4 endonuclease V
encapsulated in liposomes. Liposomes were prepared from
phosphatidylcholine/dicetyl phosphate (PC/DCP), phosphatidyl-
ethanolamine/dicetyl phosphate (PE/DCP), and phosphatidylgly-
cerol/dicetyl phosphate (PG/DCP) in 7:3 molar ratios in the manner
described in the examples below. The liposomes were added to
duplicate mixtures of W and unirradiated plasmids, the second
mixture cont~ining 1% Triton X-100 (Trademark) to dissolve the
liposomes. After incubation the mixtures were electrophoresed in
0.8% agarose to separate the plasmid forms. Lane 1 contains the
plasmid mixture untreated with liposomes. The lowest band is
UV-supercoiled plasmid and the next lowest band is
unirradiated-supercoiled plasmid. PC/DCP liposomes were added to
the mixtures in lanes 2 and 3, of which lane 3 contains Triton
X-100 (Trademark). Undissolved liposomes in lane 2 had no effect
on the W-supercoiled plasmid while dissolved liposomes in lane 3
incised the W -supercoiled plasmid and caused it to migrate in the
relaxed form in the third band from the bottom, while migration of
the unirradiated supercoiled plasmid was unaffected. PE/DCP
liposomes were added
P~'
-17- 1 3 3 8 6 4 3
to the mixtures in lanes 4 and 5, and PG/DCP liposomes were added
to the mixtures in lanes 6 and 7. Triton X-100 was present only
in lanes 5 and 7. In each case addition of Triton X-100 revealed
UV-specific incising activity of the endonuclease V trapped inside
the liposomes.
Figure 3 is a photograph of liposomes containing T4
endonuclease V which have been immunofluorescently stained.
Liposomes composed of phosphatidylcholine and cholesterol (9:1
molar ratio) and T4 endonuclease V were dried on a glass slide and
fixed with ice-cold acetone. The slide was blocked with 1% bovine
serum albumin and stained with rabbit anti-T4 endonuclease V IgG
antibodies, and goat anti-rabbit antibodies conjugated to AlkA1;ne
phosphatase. The sites of antibody binding were revealed by
incubation with 4-methylumbelliferyl phosphate (4-MUP) which was
cleaved by the Alk~l;ne phosphatase to a fluorescent dye and
visualized by fluorescent microscopy. The bright circles of
various sizes are stained liposomes containing T4 endonuclease V.
Figure 4 shows survival of XP12BE cells UV-irradiated and
treated either with (closed circles) or without (open circles)
liposomes containing endonuclease V. In panel A, cells were
treated with DPPC/PC/Chol liposomes at 0.1 ug/ml endonuclease V,
and in panel B, cells were treated with PC/DCP/Chol liposomes at
0.075 ug/ml endonuclease V.
Figure 5 shows the pH sensitivity of liposomes composed of
phosphatidylcholine, phosphatidylethanolamine, oleic acid and
cholesteryl hemisuccinate. 8-amino napthalene-1,3,6-trisulfonic
acid (ANTS) and p-xylene-bis-pyridinium bromide (DPX) were
encapsulated in liposomes composed of phosphatidylcholine,
phosphatidylethanolamine, oleic acid and cholesteryl hemisuccinate
in a 2:2:1:5 molar ratio. The liposomes were diluted into a pH 5
or pH 8 buffer, incubated at 37C and the fluorescence compared
over a 30 minute period with liposomes dissolved with Triton
X-100. ANTS is a dye whose fluorescence is quenched when a high
concentration of DPX is entrapped in liposomes. Dissolving the
liposomes with Triton X-100 dilutes the DPX relative to the ANTS
-- 1 3 3 8 6 4 3
-18-
and thus increases the fluorescence. Incubation of the liposomes
at pH 8 did not change their fluorescence (closed circles).
Incubation of the liposomes at pH 5 (open circles) increased their
fluorescence over time, demonstrating the pH sensitivity of the
liposomes.
Figure 6 shows unscheduled DNA synthesis in normal human
epidermal keratinocytes irradiated with UV-C and either untreated
or treated with liposomes containing endonuclease V. Cells were
grown on slides, either UV-irradiated or not, and incubated with
[H-3]-thymidine and with either no liposomes (open circles) or
liposomes at 0.02 ug/ml (closed circles), 0.1 ug/ml (open squares)
or 0.2 ug/ml endonuclease V (closed squares). After four hours
the cells were fixed and coated with nuclear track emulsion.
After 7 days exposure the slides were developed and grains over
the nuclei of 25 cells not heavily labeled (i.e. not in S phase)
for each slide were counted and averaged. Cells treated with
liposomes showed enhanced unscheduled DNA synthesis after
UV-irradiation compared to untreated control cells.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
As discussed above, the present invention relates to 1) a
method for purifying DNA repair enzymes through the sequential use
of a molecular sieve chromatography column and a nucleic acid
affinity column, and 2) the use of liposomes to administer DNA
repair enzymes to living cells.
The purification and administration aspects of the invention
can be applied to a variety of DNA repair enzymes now known or
subsequently developed or discovered. In particular, the
invention can be used with phage T4 endonuclease V,
O -methylguanine-DNA methyltransferases, and with the other DNA
repair enzymes discussed and listed above.
With regard to the purification aspects of the invention, the
first step of the purification process -- molecular sieving --
serves to separate the DNA repair enzymes from the vast majority
of proteins with larger sizes based on relative rates of migration
-19- 1 338643
of the DNA repair enzymes and the contaminating proteins through
the molecular sieve matrix.
Molecular sieving can be accomplished by many methods,
including gel filtration and electrophoresis. In gel filtration
proteins flow around and through pores in beads made from dextran,
polyacrylamide, agarose, agarose and acrylamide composites, or
other materials. The size of the bead pores include or exclude
proteins based on size. In electrophoresis, proteins move in an
applied electric field through a sizing matrix.
The preferred molecular sieving method for use with the
present invention is gel filtration because the enzyme can be
easily recovered and because the method is independent of such
factors as net protein charge. The pore size of the beads used
with this method are selected to q~; ;7e separation of DNA repair
enzymes from the bulk of other proteins. A general guideline for
selecting the gel filtration matrix is that the gel should have an
exclusion limit greater than about twice the molecular weight or
Stokes' radius of the DNA repair enzyme and less than about 60,000
daltons or 35 Angstroms.
A wide variety of elution buffers may be used to elute the
DNA repair enzyme from the gel filtration column. The selected
buffer should satisfy the following criteria: 1) the buffer
should not denature or inactivate the DNA repair enzyme, 2) the
buffer should not permit ionic adsorption of the DNA repair enzyme
to the gel filtration media, and 3) the buffer should be
compatible with loading of the eluate onto the nucleic acid
affinity column, that is, the elution buffer should be chosen so
that complexes will form between the DNA repair enzyme and the
immobilized nucleic acids of the affinity column.
The second step of the purification process -- nucleic acid
binding -- separates the DNA repair enzymes from the rl ~;n;ng
protein impurities by the ability of DNA repair enzymes to
reversibly bind to nucleic acids. Separation by nucleic acid
binding can be accomplished by various methods, including nucleic
acid affinity chromatography. In this method, nucleic acids are
-20- 1 3 3 8 6 4 3
immobilized on an inert matrix, such as agarose, polyacrylamide
beads, cellulose or other media. Depending on the DNA repair
enzyme which is being purified, the immobilized nucleic acids may
be double- or single-stranded DNA, double- or single-stranded RNA,
or other types, lengths, structures or combination of nucleic
acids, such as tRNA, Z-DNA, supercoiled DNA, ultraviolet-
irradiated DNA or DNA modified by other agents. Single-stranded
DNA is in general preferred.
The nucleic acids may be attached to the solid phase matrix
by a variety of methods, including covalent attachment of the
nucleic acid through primary amines or by adsorbing the nucleic
acids to a matrix such as cellulose, which releases nucleic acids
slowly. The preferred immobilization method is to use
cyanogen-bromide activated Sepharose (Trademark) and to bind the
nucleic acids to the activated Sepharose (Trademark) covalently.
Alternatively, single-stranded DNA covalently bound to agarose can
be purchased commercially from Bethesda Research Labs,
Gaithersburg, Maryland (Catalog No. 5906SA).
The DNA repair enzymes are applied to the nucleic acids in a
solution which should satisfy the following criteria: 1) the
solution should permit reversible binding of the DNA repair enzyme
to the nucleic acids, 2) the solution should reduce non-specific
binding of contaminating proteins to the nucleic acids, and 3) the
solution should not cause damage to the nucleic acids. In
general, a neutral buffered solution with physiological saline and
1 mM EDTA will satisfy these criteria. As discussed above, in
accordance with the invention, the elution fractions from the
molecular sieve column are applied directly to the nucleic acid
affinity column. Accordingly, the elution buffer used with the
molecular sieve column should be chosen to satisfy the foregoing
criteria.
The bound DNA repair enzymes are eluted from the nucleic acid
affinity column with a gradient which removes the enzyme from the
nucleic acid at a characteristic condition and concentrates the
enzyme by the focusing effect of the gradient. The elution
-21- 1 338643
system, however, should not denature the enzyme or introduce
cont ;n~nts into the final product. A gradient of NaCl up to 1.0
M will in general be sufficient to reverse binding of most DNA
repair enzymes to nucleic acids. In appropriate cases, the
gradient may be one of another salt, increasing or decreasing pH,
temperature, voltage or detergent, or, if desired, a competing
ligand may be introduced to replace the nucleic acid binding.
With regard to the administration aspects of the invention,
the liposomes which are used to administer the DNA repair enzymes
can be of various types and can have various compositions. The
primary restrictions are that the liposomes should not be toxic to
the living cells and that they should deliver their contents into
the interior of the cells being treated.
The liposomes may be of various sizes and may have either one
or several membrane layers separating the internal and external
compartments. The most important elements in liposome structure
are that a sufficient amount of enzyme be sequestered so that only
one or a few liposomes are required to enter each cell for
delivery of the DNA repair enzyme, and that the liposome be
resistant to disruption. Liposome structures include small
nll~llar vesicles (SUVs, less than 250 angstroms in diameter),
large llnil ?llar vesicles (LUVs, greater than 500 angstroms in
diameter), and multilamellar vesicles (MLs). In the examples
presented below, SUVs are used to a~' ~ni ster DNA repair enzymes.
SUVs can be isolated from other liposomes and unincorporated
enzyme by molecular sieve chromatography, which is precise but
time consuming and dilutes the liposomes, or differential
centrifugation, which is rapid but produces a wider range of
liposome sizes.
The liposomes may be made from natural and synthetic
phospholipids, glycolipids, and other lipids and lipid congeners;
cholesterol, cholesterol derivatives and other cholesterol
congeners; charged species which impart a net charge to the
membrane; reactive species which can react after liposome
formation to link additional molecules to the liposome membrane;
-
-22- l 3386~3
and other lipid soluble compounds which have chemical or
biological activity.
Liposome membranes undergo a phase transition from
crystalline to liquid at a temperature (Tc) characteristic of the
phospholipid composition. When the phospholipid is heated above
Tc and then cooled, the membrane retains water in its amphiphilic
lattice and has the characteristics of a gel. In order to achieve
the liquid or gel state, the phospholipid composition must be such
that the Tc is lower than the temperature which inactivates the
entrapped enzyme. Cholesterol in the phospholipid mix effectively
reduces the Tc by broadening the range at which phase transition
occurs. In view of these requirements, a suitable mixture for
preparing the liposomes of the present invention comprises
phosphotidyl choline (or a derivative thereof with a Tc of less
than 42C), dicetyl phosphate (or a negatively charged species at
neutrality), and cholesterol (or a cholesterol derivative) at a
molar ratio of 7:2:1.
As discussed above, pH sensitive liposomes are a preferred
type of liposome for use with the present invention. As described
by Robert Straubinger, Keelung Hong, Daniel Friend and Demetrios
Papahadjopoulos in their paper entitled "Endocytosis of Liposomes
and Intracellular Fate of Encapsulated Molecules: Encounter with a
Low pH Compartment after Internalization in Coated Vesicles,"
referred to above, one pathway for the entry of liposomes into
cellular cytoplasm is by endocytosis into lysozymes of low pH.
Accordingly, liposomes which are stable at neutral pH but release
their contents at acidic pH can be used to deliver enzymes into
the lysozymes of the cytoplasm, whereupon the contents are
released. Since DNA repair enzymes like the T4 endonuclease V are
relatively stable at low pH, this method allows efficient delivery
of active enzymes into cells.
Liposomes can be made sensitive to the low pH of the
lysozymes by the lipid composition. See generally chapter 11 of
the book CELL FUSION, referred to above. In particular, pH
sensitive liposomes can be prepared by using phospholipids which
-23- l 338643
form lipid bilayers when charged but fail to stack in an ordered
fashion when neutralized. An example of such a phospholipid is
phosphatidylethanolamine, which is negatively charged above pH 9.
The net charge of a phospholipid can be maintained at a pH which
would otherwise neutralize the head groups by including charged
molecules in the lipid bilayer which themselves can become
neutralized. Examples of these charged molecules are oleic acid
and cholesteryl hemisuccinate, which are negatively charged at
neutral pH but become neutralized at pH 5. The effect of
combining these together in a lipid bilayer is that at pH 9 all
molecules are charged; at pH 7 the net negative charge of the
oleic acid and cholesteryl hemisuccinate maintains the stability
of the phosphatidylethanolamine, and at pH 5 all components are
protonated and the lipid membrane is destabilized. Additional
neutral molecules, such as phosphatidylcholine, can be added to
the liposomes as long as they do not interfere with stabilization
of the pH sensitive phospholipid by the charged molecules.
The examples presented below illustrate two particular
methods for producing pH sensitive liposomes. First, the
combination of phosphatidylethanolamine and cholesteryl
hemisuccinate (CHEMS) in the lipid membrane destabilizes the
liposome at a pH of less than 4.5, as described by Joe Bentz,
Harma Ellens and Francis Szoka in their paper entitled
"Destabilization of Phosphatidylethanolamine-Cont~;ning Liposomes:
Hexagonal Phase and Asymmetric Membranes", referred to above.
This paper measured destabilization by a lowering in the phase
transition temperature or by the leakage of one liposome in the
presence of another liposome of different composition. See also
Harma Ellens, Joe Bentz and Francis C. Szoka, "pH-Induced
destabilization of phosphatidylethanolamine-cont~in;ng liposomes:
role of bilayer contact," referred to above. Second, the
inclusion of oleic acid with phosphatidylethanolamine also
destabilizes the lipid bilayer at a pH of less than 6.5, and
imparts a net negative charge to the liposome at neutral pH, as
discussed in "pH-Sensitive Liposomes Mediate Cytoplasmic Delivery
-24- 1338643
of Encapsulated Macromolecules" by Robert Straubinger, Nejat
Duzgunes and Demetrios Papahadjopoulos, referred to above.
The examples also illustrate that liposomes composed of a
mixture of phosphatidylcholine and phosphatidylethanolamine are
more pH sensitive than those composed of phosphatidylethanolamine
alone. Further, liposomes in which the molar ratio of CHEMS to
the r~_q;ning components of the liposome is about 1:1 were found
to respond to pH changes faster than liposomes containing lesser
amounts of CHEMS, e.g., 20 minutes versus three hours.
Accordingly, a preferred composition for the pH sensitive
liposomes is phosphatidylethanolamine, phosphatidylcholine, oleic
acid, and cholesteryl hemisuccinate (PE/PC/OA/CHEMS) in a molar
ratio of 2:2:1:5. Of course, other compositions for producing pH
sensitive liposomes now known or subsequently developed can be
used in the practice of the invention.
The liposomes of the present invention are prepared by
combining a phospholipid component with an aqueous component
containing the DNA repair enzyme under conditions which will
result in vesicle formation. The phospholipid concentration must
be sufficient to form lamellar structures, and the aqueous
component must be compatible with biological stability of the
enzyme. Methods for combining the phospholipid and aqueous
components so that vesicles will form include: drying the
phospholipids onto glass and then dispersing them in the aqueous
component; injecting phospholipids dissolved in a vaporizing or
non-vaporizing organic solvent into the aqueous component which
has previously been heated; and dissolving phospholipids in the
aqueous phase with detergents and then removing the detergent by
dialysis. The concentration of the DNA repair enzyme in the
aqueous component can be increased by lyophilizing the enzyme onto
dried phospholipids and then rehydrating the mixture with a
reduced volume of aqueous buffer. SUVs can be produced from the
foregoing mixtures either by sonication or by dispersing the
mixture through either small bore tubing or through the small
orifice of a French Press.
-25- 1 338643
In the examples presented below, SUVs were prepared by drying
phospholipids onto glass, rehydrating them in aqueous buffer
cont~;n;ng the DNA repair enzyme with shaking at 37C, sonicating
the resulting mixture, and isolating the SUVs containing the DNA
repair enzyme by molecular sieve chromatography and concentrating
the SUVs by centrifugation. Figure 3 illustrates the success of
this technique for incorporating DNA repair enzymes into
liposomes.
DNA repair enzymes incorporated into liposomes can be
~1 ;n;stered to living cells internally or topically. Internal
administration to animals or humans requires that the liposomes be
pyrogen-free and sterile. To eliminated pyrogens, pyrogen-free raw
materials, including all chemicals, enzymes, and water, are used
to form the liposomes. Sterilization can be performed by
filtration of the liposomes through 0.2 micron filters. For
injection, the liposomes are suspended in a sterile, pyrogen-free
buffer at a physiologically effective concentration. Topical
~l~ ;nl~tration also requires that the liposome preparation be
pyrogen-free, and sterility is desirable. In this case, a
physiologically effective concentration of liposomes can be
suspended in a buffered polymeric glycol gel for even application
to the skin. In general, the gel should not include non-ionic
detergents which can disrupt liposome membranes. Other vehicles
can also be used to topically ~.' ;n;~ter the liposomes. The
concentration of the enzyme in the final preparation can vary over
a wide range, a typical concentration being on the order of 50
ug/ml. In the case of pH sensitive liposomes, lower
concentrations of the DNA repair enzyme can be used, e.g., on the
order of 0.01 to 1.0 ug/ml for liposomes administered to cells
internally. In case of topical application, higher liposome
concentrations are used, e.g., ten or more times higher.
General discussions of liposomes and liposome technology can
be found in an article entitled "Liposomes" by Marc J. Ostro,
published in SCIENTIFIC AMERICAN, January 1987, volume 256, pages
102-111, and in a three volume work entitled LIPOSOME TECHNOLOGY,
1 338643
-26-
edited by G. Gregoriadis, 1984, published by CRC Press, Boca
Raton, Florida.
The topical administration of liposome encapsulated DNA
repair enzymes has been considered until now, but it is recognized
that this invention has a more general application in the topical
delivery to living skin of a wide variety of biologically active
proteins to achieve a biological effect.
Many human diseases result from dysfunctional skin
metabolism, either because skin cells fail to perform a function,
function improperly, or overproduce a function. Examples of such
diseases are xeroderma pigmentosum (absence of DNA repair enzyme
in skin), some forms of albinoism (absence of tyrosinase in
melanocytes), and psoriasis (overproduction of cytokines such as
IL-l). Other skin disease may respond to the intracellular
delivery of biologically active proteins, such as melanomas which
may respond to repressors of oncogene function (rb gene product).
The common theme of the therapy for these diseases is the delivery
of bioactive materials across stratum corneum and then across the
outer cellular membrane into the internal space of living skin
cells. The present invention can accomplish this not only for the
delivery of DNA repair enzymes, but also for the delivery to skin
of other biologically active proteins encapsulated in liposomes.
An important distinction is made here between liposomes
encapsulating small molecular weight drugs for extracellular
release, and the present invention, which provides intracellular
delivery of large molecular weight, biologically active proteins.
It is also important to note that the conventional wisdom is that
only very small molecules, which includes many drugs but excludes
all proteins, penetrate the stratum corneum, and that proteins
alone cannot cross the outer cell membrane, other than a few very
specialized receptor-binding proteins. The present invention
demonstrates that proteins can be delivered across the outer skin
layer and into living cells, without receptor binding, by liposome
encapsulation.
~,',~,
-
-26a- 1 338643
The method for producing topically applied liposomes
encapsulating biologically active proteins is exemplified by the
procedure for encapsulation and administration of DNA repair
enzymes. The biologically active protein is preferably
electrophoretically pure. Also, it should be encapsulated under
conditions which are gentle and do not inactivate the protein's
biological activity. The concentration of liposomes necessary for
topical administration can be determined first by measuring the
biological effect of the protein in liposomes on target skin cells
in culture. Once the optimal active range is found, equal or
greater concentrations are formulated in a lotion or gel for even
application to skin.
Without intending to limit it in any manner, the present
invention will be more fully described by the following examples.
EXAMPLE 1
Purification of Endonuclease V
(1) Enzyme purification.
(A) Cell growth and induction. The E. coli strain
SR1268 harboring the plasmid pTACdenV is described by J.
Chenevert, L. Naumovski, R.A. Schultz and E.C. Friedberg,
MOLECULAR AND GENERAL GENETICS, 1986, Vol. 203, pages 163-171. A
sample of this strain was obtained from Dr. Errol Friedberg,
Department of Pathology, Stanford University, School of Medicine,
Stanford, California 94305. A culture of the bacteria was
prepared in 200 ml LB+amp (10 g/l tryptone, 5 g/l yeast extract,
10 g/l NaCl, 125 ug/ml ampicillin, pH 7.5) and incubated overnight
at 37C. The culture was diluted to 2 liters in LB+amp and
incubated at 37C until the optical density at 600 nm was 0.3.
Isopropyl-beta-D-thiogalactopyranoside was added to 1 mM and the
incubation continued for 60 min.
(B) Cell lysis and protein precipitation. The cells
were collected by centrifugation at 12,785 x g for 15 min at 4C,
and were resuspended in 200 ml of TES (50 mM Tris, pH 8.0, 50 mM
Na2EDTA, 15% sucrose). 0.2 g of lysozyme was added and the
mixture was incubated at 25C, for 30 min. 160 ml ice cold
-26b- 1 3 3 8 6 4 3
distilled water and 40 ml 10% streptomycin sulfate were then
added, and stirred at 4C for 30 min. The lysate was centrifuged
at 15,188 x g for 15 min. and the supernatant was collected. 83.2
g of ammonium sulfate was slowly added with stirring at 4C, and
stirring was continued for 30 min. The mixture was centrifuged at
15,188 x g for 15 min, and the supernatant was collected. 79.6 g
ammonium sulfate was added and stirred at 4C for 30 min. The
mixture was centrifuged at 15,188 x g for 15 min, and the
precipitate was resuspended in Buffer A (50 mM Tris, pH 8, 50 mM
~k
-27- 1 3 3 8 6 4 3
NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl
fluoride) and dialyzed against 1.0 1 Buffer A overnight at 4C.
(C) Gel filtration. The dialyzed proteins were loaded
on a standard column (2.5 cm d. x 30 cm) of AcA54 gel filtration
media (LKB) at a flow rate of 50 ml/hr. The proteins were eluted
isocratically with Buffer A, and fractions of 5 ml were monitored
for optical density at 280 nm. The second large peak of protein,
which elutes at approximately R(f)=1.67, was pooled.
(D) Single-stranded DNA chromatography. Single-
stranded DNA Sepharose was prepared by boiling calf thymus DNA andcovalently attaching it to CNBr-activated Sepharose (Pharmacia)
according to the manufacturer's instructions. This was packed in
a Superflo (Trademark) 50 radial flow column (Sepragen).
Alternatively, the single-stranded DNA/agarose column produced by
Bethesda Research Labs was used (see above). The second peak of
protein from gel filtration was loaded directly onto the
single-stranded DNA column at a flow rate of 400 ml/hr and washed
with 250 ml of Buffer A. At the same flow rate the column was
developed with a 200 ml linear gradient of 50 mM to 1.0 M NaCl in
Buffer A. Fractions of 5 ml were monitored for optical density at
280 nm. The peak of protein which elutes at about midway in
gradient development was pooled. As demonstrated below, this peak
was pure endonuclease V.
(E) Storage. Herring sperm DNA was added to the enzyme
to a concentration of 20 ug/ml and the resulting mixture was
dialyzed overnight at 4C against a buffer of 50 mM Tris, pH 8.0,
100 mM NaCl, 10 mM EDTA, 1 mM dithiothreitol, 0.1 mM
phenylmethylsulfonyl fluoride. Polyethylene glycol was added to
the mixture at a concentration of 3% and the final solution was
stored at 4C until used.
(2) Assay for endonuclease V activity.
The assay used to measure endonuclease V activity measured
the relaxation by the enzyme of supercoiled plasmid DNA produced
by single-stranded breaks introduced in UV-irradiated DNA. A unit
of enzyme activity is defined as the amount of enzyme which will
produce on the average one break per molecule in 1 ng of pBR322
1 338643
-28-
plasmid irradiated with 40 J/m of 254 nm UV in a 10 min.
incubation at 37C.
(A) Substrate. Plasmid pBR322 was irradiated with 40
J/m of 254 nm UV and mixed with unirradiated plasmid pSV2neo.
Because the pBR322 plasmid is smaller than the pSV2neo plasmid,
both its supercoiled and relaxed forms migrate faster in neutral
agarose gels than the corresponding forms of pSV2neo. However,
the supercoiled form of pSV2neo migrates faster than the relaxed
form of pBR322. The substrate for the assay is the W -irradiated
pBR322 DNA and the pSV2neo DNA serves as a control for
non-specific nuclease activity. The substrate DNA was prepared at
20 ug/ml of each plasmid in Endo V Buffer (50 mM NaHP04, 100 mM
NaCl, 10 mM EDTA, 1 mM DTT, 0.1 ug/ml bovine serum albumin, pH
6.5).
(B) Reaction. 25 ul of substrate DNA was mixed with 25
ul of enzyme preparation from step l(E) above, diluted in Endo V
Buffer, and incubated at 37C for 10 min. The reaction was
stopped by addition of 5 ul lOX Gel Loading Buffer (0.25%
bromophenol blue, 30% Ficoll (Trademark)).
(C) Agarose gel electrophoresis. A 0.8% agarose gel
was prepared in TAE buffer (40 mM Tris, pH 8.4, 5 mM sodium
acetate, 1 mM EDTA). 10 ul of each reaction was loaded and
electrophoresed at 7 V/cm. When the dye had migrated 2/3 of the
gel length, the gel was soaked in ethidium bromide (1 ug/ml),
destained in 1 mM MgS04, and viewed by transillumination with 360
nm W . The gel was photographed using a red Wrattan (Trademark)
filter and Polaroid (Trademark) positive/negative film Type 665.
The negative was washed in distilled water.
(D) Film analysis. For quantitation of endonuclease
activity, the negative was scanned by a densitometer and the
analog voltage readings were stored in digital form in a computer.
Labtech Chrom (Trademark) software (Laboratory Technologies
Corporation, Wilmington, Massachusetts) was used to integrate the
area of each band detected by densitometry. The average number of
breaks in each plasmid was calculated from the natural log of the
fraction
~'
. . ,
_ -29- l 3 3 8 6 4 3
of supercoiled molecules. The average number of UV-specific
breaks was calculated from the difference in average breaks
between pBR322 and pSV2neo DNA, corrected for the difference in
untreated samples.
(3) Results.
Typical results from the purification process of the
invention were as follows: Two liters of bacterial culture were
prepared and lysed. After the precipitation of nucleic acids by
streptomycin sulfate, 660 mg of protein remained. 525 mg of
protein were recovered from ammonium sulfate precipitation and
loaded onto the gel filtration column, and 15 mg of pure
endonuclease V were recovered after the DNA affinity column at a
concentration of 0.2 mg/ml. The enzyme activity was 10,000 units
per ug protein. The preparation produced a single band on
polyacrylamide gel electrophoresis (see Figure 1) and only a
single band appeared on Western blots of protein probed with
rabbit antiserum to endonuclease V. The enzyme was stable for at
least four months at 4C, at least 5 days at 37C, and had a half
life at 42C of greater than 30 min.
EXAMPLE 2
Purification of O -Methylguanine-DNA Methyltransferase
O -methylguanine-DNA methyltransferase is a DNA repair enzyme
which removes alkyl groups from DNA and transfers them to itself
in a suicide reaction. The properties of this enzyme have been
summarized by Daniel Yarosh, in a review entitled "The role of
O -methylguanine-DNA methyltransferase in cell survival,
mutagenesis and carcinogenesis", published in MUTATION RESEARCH,
volume 145, pages 1-16, 1985.
(1) Enzyme purification.
(A) Cell growth and induction. E. coli strain N445
harboring the plasmid pSM31 was grown to stationary phase in
LB+amp, as described above in Example 1. The plasmid pSM31
contains the entire ada gene with its own promoter, and codes for
the _ coli O -methylguanine-DNA methyltransferase. A sample of
the N445 strain was obtained from Dr. Sankar Mitra, Biology
-
-30- l 3 3 8 6 4 3
Department, Oak Ridge National Laboratory, Oak Ridge, Tennessee
37831. The production of the transferase by these cells was
induced by adding N-methyl-N'-nitro-N-nitrosoguanidine to a
concentration of 0.5 ug/ml, and incubating the culture at 37C for
90 min.
(B) Cell lysis and protein precipitation. The cells
were collected and lysed by the same methods as described in
Example 1. Nucleic acids were precipitated by streptomycin
sulfate as described in Example 1. Ammonium sulfate was added at
112 g per 200 ml supernatant, and stirred at 4C for 60 min. The
precipitated proteins were collected by centrifugation at 15,188 x
g for 30 min, resuspended in 5 ml of Buffer A, and dialyzed
overnight against 1.0 1 Buffer A at 4C.
(C) Gel filtration and single-stranded DNA
chromatography. Chromatography of the cell lysate was performed
as described for endonuclease V. The transferase eluted from the
AcA54 gel as a shoulder on the first large optical density peak,
and from single-stranded DNA after about one-third of the
gradient. As demonstrated below, the pooled peak was pure
O -methylguanine-DNA methyltransferase.
(2) Assay for O -methylguanine-DNA methyltransferase
activity.
The assay used to measure the activity of the
O -methylguanine-DNA methyltransferase enzyme measured the
transfer of radiolabeled methyl groups from DNA to protein, and
has been described by B. Myrnes, K. Nordstrand, K. Giercksky, C.
Sjunneskog and H. Krokan in a paper entitled "A simplified assay
for O -methylguanine-DNA methyl transferase and its application to
human neoplastic and non-neoplastic tissues," published in
CARCINOGENESIS, 1984, volume 5, pages 1061-1064.
(A) Substrate. The substrate for the assay was DNA
alkylated with a simple methylating agent and then enriched for
O -methylguanine by depurinating other alkylated purines. Calf
thymus DNA at 5 mg/ml was reacted with methylnitrosourea
containing tritium in the methyl moiety at 1 mCi per ml of a 0.2 M
-31- 1 3 3 8 6 4 3
sodium cacodylate buffer, pH 7, 5 mM EDTA, at 37C for 4 hr. The
DNA was precipitated by ethanol, washed with ethanol, and
resuspended at 2.5 mg/ml in 0.1 M NaCl, 10 mM sodium citrate, 10
mM potassium dihydrogen phosphate, pH 7.4. The DNA was heated at
80C for 16 hrs., ethanol precipitated, washed and resuspended in
10 mM Tris, pH 7, 1 mM EDTA. This substrate contained more than
half of all labeled adducts as 0 -methylguanine.
(B) Reaction. 0.33 pmol of 0 -methylguanine in DNA was
mixed with the enzyme preparation in transferase buffer of 70 mM
Hepes, pH 7.1, 1 mM EDTA, and incubated at 37C for 30 min to
allow transfer of labeled adducts from DNA to protein. The
mixture was brought to 5% trichloroacetic acid and heated at 80C
for 30 min to precipitate proteins and solubilize unreacted
0 -methylguanine. The precipitated proteins were separated from
solubilized bases by filtering through glass fiber filters. The
filters were washed with ethanol and the trapped radioactivity
measured by scintillation counting. The amount of transferase was
calculated from the trapped radioactivity and the known specific
activity of the labeled methylated bases.
(3) Results.
Typical results from this purification were as follows: After
precipitation of nucleic acids from 2.0 1 of cell extract, 218 mg
of protein were loaded on the gel filtration column, 77 mg of
protein were collected and loaded on the single-stranded DNA
column, and 14 mg of pure 0 -methylguanine-DNA methyltransferase
were recovered. Enzyme activity was 3,000 pmol methyl groups
transferred per mg protein. The protein showed a single band (see
Figure 1) on polyacrylamide gels and in Western blots using rabbit
antiserum to the transferase protein.
EXAMPLE 3
Encapsulation of denV Endonuclease V
in PC/DCP and PC/SA Liposomes
(1) Preparation of liposomes.
As discussed above, liposomes may be prepared by many methods
using many lipid and non-lipid mixtures over a broad range of
-32- 1 3 3 8 6 4 3
concentrations. The following methods were used in this example.
22 mg of egg yolk phosphatidylcholine and either 13.5 mg of
dicetyl phosphate or 7.3 mg of stearylamine were dissolved in 5 ml
of chloroform. Two mls of this mixture were dried to a film in a
25 ml round bottom flask by an air stream in a water bath at 37C.
The film was further dried by vacuum for 60 min. Two mls of denV
endonuclease V at 0.2 mg/ml prepared as described in Example 1
were added, and the mixture vortexed to dissolve the lipid in the
aqueous solution. The flask was placed in a sonication bath for
60 min. In the case of phosphatidylcholine/dicetyl phosphate
liposomes, the solution was then centrifuged at 12,000 x g for 5
min, and the supernatant drawn off and discarded. The pellet of
liposomes was resuspended in phosphate buffered saline (PBS) and
washed again by centrifugation. The pellet of liposomes was
finally resuspended in 1 ml of PBS. In the case of
phosphatidylcholine/stearylamine liposomes, the solution was
eluted through a 1.5 x 30 cm column of AcA54 gel filtration media,
and 2 ml fractions were collected. The liposomes eluted in
fractions 10-13, and these fractions were pooled. Liposome
concentration was measured by diluting the suspended liposomes
1:100 and measuring the optical density at 600 nm.
(2) Assay of liposome encapsulation.
(A) Encapsulation efficiency. A radioactive tracer
molecule was included in the aqueous protein solution, and the
percentage of radioactivity found in the liposome fraction was
compared to the radioactivity found in the remaining fraction.
(B) Endonuclease V activity. The endonuclease V
activity assay described in Example 1 was used to measure active
enzyme in the liposomes. The liposome preparation was added to
duplicate assays, one of which contained 1% Triton X-100 to
dissolve the liposomes. Comparison of the activity between intact
and dissolved liposomes served as a measure the amount of active
enzyme entrapped in liposomes. Figure 2 illustrates the type of
results achieved with this assay protocol.
--- 1 3 3 8 6 43
-33-
(3) Results.
Typical results from this method of liposome preparation were
as follows: Liposomes were prepared with phosphatidylcholine and
stearylamine. Using [H-3]-thymidine as a tracer and separation of
liposomes from unincorporated tracer by gel filtration, 42,400 cpm
were recovered in the liposome fraction and 10,939,600 cpm were
recovered from the r ~;n;ng fraction, for an encapsulation
efficiency of 0.39%. No endonuclease V activity was detected in
liposomes without Triton X-100, while dissolved liposomes
contained 23,000 units in 1 ml, or 0.59% of the initial activity
of 4,000,000 units of endonuclease V.
EXAMPLE 4
Encapsulation of denV Endonuclease V
in PC/DCP/Chol and PC/SA/Chol Liposomes
(1) Preparation of liposomes.
Liposomes were prepared from phosphatidyl choline, dicetyl
phosphate and cholesterol, or from phosphatidyl choline,
stearylamine and cholesterol, each in a 7:2:1 molar ratio, in the
manner described in Example 3 using the centrifugation technique
of Example 3 to isolate liposomes.
(2) Assay of liposome encapsulation.
(A) Enzyme concentration. Concentration of the enzyme
entrapped in liposomes was measured by enzyme-linked immunosorbent
assay (ELISA). Liposomes were diluted to an optical density at
600 nm of 1.0 in 0.1 ml PBS and 25 mM octyl-beta-D-galac-
topyranoside to dissolve the liposomes. Fifty ul was then diluted
in duplicate into 0.2 ml coating buffer (50 mM sodium bicarbonate,
pH 9.6, 0.1 mg/ml thimersol) and serially diluted 1:1 down columns
of wells in a 96-well microtiter plate. Standards of purified
endonuclease V at 5 ug/ml in PBS/octylgalactopyranoside were
identically prepared. After overnight incubation at 4C, the
wells were washed with 50 mM Tris, pH 8, 150 mM NaCl (TBS) + 0.1%
non-idet NP 40 (Trademark) detergent (TBS/NonI), and blocked with
0.2% bovine serum albumin in coating buffer for 2 hours at 25C.
The wells were washed and primary antiserum of rabbit
anti-endonuclease V
t
~,r~f
-34- 1 3 3 8 6 4 3
IgG antibodies (5 ug/ml) were added for 2 hours at 25C. The
wells were washed and secondary anti-serum of goat anti-rabbit IgG
antibodies conjugated to ~lkAline phosphatase were added for 30
minutes at 25C. The wells were washed and o-nitrophenyl
phosphate (1 mg/ml) was added. After 30 min. incubation, the
optical densities of the wells were measured at 405 nm. The
concentration of enzyme in the liposome preparation was calculated
from a standard curve of optical density versus enzyme
concentration for the endonuclease V standards.
(B) Endonuclease V activity. Endonuclease V activity
was determined in the same manner as in Example 3.
(3) Results.
The results of these experiments are shown in Table I. As
shown therein, the PC/DCP/Chol liposomes and the PC/SA/Chol
liposomes incorporated similar amounts of enzyme in terms of
ug/ml. In terms of enzyme activity, however, the PC/SA/Chol
liposomes exhibited over four times the activity of the
PC/DCP/Chol liposomes.
Liposomes were prepared using other phospholipids including
distearoylphasphatidyl choline (DSPC), dimyristoylphosphatidyl
choline (DMPC), dipalmitoylphosphatidyl choline (DPPC),
phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), and
the results with these are also shown in Table 1. The activity of
the encapsulated enzyme for these other liposomes was verified
qualitatively by visual ex; In~tion of the activity gel, but not
measured quantitatively.
EXAMPLE 5
Enhancement of Human DNA Repair
by Liposomes Containing Endonuclease V
(1) Growth and irradiation of human cells in culture.
Human cells were grown using standard tissue culture methods
and were used for measuring the enhancement of DNA repair by
liposomes containing endonuclease V. The human cells used were:
secondary culture of normal human epidermal keratinocytes, normal
human fibroblast line WI-38, and SV-40 transformed fibroblast line
1 338643
-35-
XP12BE from a patient with xeroderma pigmentosum. All cells were
incubated at 37C in a humidified 5% C02 atmosphere attached to
plastic dishes. The normal human epidermal keratinocytes were
purchased from and cultured according to the directions of
Clonetics Corporation, San Diego, Calif., in Keratinocyte Growth
Media, which is a modified MDCB-151 media supplemented with bovine
pituitary extract. The remaining cells were cultured in Dulbecco's
;n; ~1 essential media with 10% newborn calf serum, supplemented
with antibiotics and vitamins. All cells were grown to near
confluence and then subcultured at a 1:4 ratio. For irradiation,
the cells were drained of all media, and exposed to a germicidal
UV lamp without the dish lid. The lamp output was predominately
at 254 nm and the fluence rate was measured by a UVX (Trademark)
digital radiometer from Ultra-violet Products, Inc., San Gabriel,
Calif., equipped with the UVX-25 probe for 254 nm light. The
fluence rate was either 1 J/m /sec or 2.5 J/m /sec in all
experiments.
(2) Assays for enhanced DNA repair.
(A) Alkaline agarose gel assay for liposome-mediated
incision of W -irradiated DNA. The theoretical basis and
practical application of the AlkA11ne agarose gel assay for
single-stranded breaks is described in a paper authored by Steven
E. Freeman, Anthony D. Blackett, Denise C. Monteleone, Richard B.
Setlow, Betsy M. Sutherland and John C. Sutherland entitled
"Quantitation of Radiation-, Chemical-, or Enzyme-Induced Single
Stranded Breaks in Nonradioactive DNA by Alkaline Gel
Electrophoresis: Application to Pyrimidine Dimers", published in
ANALYTICAL BIOCHEMISTRY, volume 158, pages 119-129, 1986. Human
cells were irradiated with 100 J/m of 254 nm W, and then media
containing liposomes was incubated with the cells for 2 hrs. The
media was removed, the cells were scraped from the dish into PBS,
and the DNA purified. An aliquot of DNA was electrophoresed at 3
V/cm in a 0.4% agarose gel and stained with 1 ug/ml ethidium
bromide. The gel was photographed and the image of the gel lanes
on the developed film negative was scanned by a densitometer. The
output of the densitometer was converted from analog voltages to
~',.
.. ~ . j
-36- 1 3 3 8 6 4 3
digital values and stored in a computer file. DNA molecular
weight markers were included in the gel and also scanned. The
number average molecular weight of the DNA in each lane was
calculated as described by Freeman et al. by calibrating mobility
in the gel with the molecular weight markers. Liposome-mediated
incision resulted in smaller number average molecular weight DNA
than DNA extracted from irradiated cells not treated with
liposomes or unirradiated DNA treated with liposomes.
(B) DNA repair replication. Repair of DNA includes
resynthesis of damaged DNA excised during repair. Incorporation of
radioactive DNA bases during this repair synthesis results in
radioactive high molecular weight DNA in cells undergoing repair.
The use of this measure of DNA repair synthesis is reviewed in the
1974 ANNALS OF INTERNAL MEDICINE paper on xeroderma pigmentosum by
J. Robbins et al., referred to above. In the standard method for
measuring repair synthesis, human cells were incubated with 0.01
uCi/ml [C-14] thymidine to uniformly label their DNA and then
incubated with 10 mM hydroxyurea for 60 min. to suppress normal
DNA synthesis. The cells were then irradiated with 100 J/m 254
nm UV, and incubated with 10 mM hydroxyurea, liposomes containing
endonuclease V and [H-3]-thymidine at 5 uCi/ml for 4 hrs, during
which time repair synthesis occurred. The media was removed, and
the cells were scraped into PBS, collected on glass fiber filters,
then lysed and washed with 5% trichloroacetic acid and ethanol.
Unincorporated [C-14]- and [H-3]-thymidine and small molecular
weight DNA were washed from the filters, but high molecular weight
DNA was precipitated and remained. The filters were dried and the
radioactivity measured by scintillation counting. The ratio of
[H-3]- to [C-14]-thymidine incorporated into DNA was used as a
measure of the amount of repair DNA synthesis per unit DNA. Repair
synthesis was normalized to 100% for irradiated samples not
treated with liposomes.
(C) Cell survival. Survival of cells following UV
irradiation was measured by metabolism of a tetrazolium salt as
described by Tim Mosmann in a paper entitled "Rapid Colorimetric
_37_ 1 3 3 8 6 4 3
Assay for Cellular Growth and Survival: Application to
Proliferation and Cytotoxicity Assay", published in the JOURNAL OF
IMMUNOLOGICAL METHODS, volume 65, pages 55-63, 1983. Living cells
metabolize the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide), which is yellow, to
MTT-formazan, which is blue. The formazan can be measured by a
multiwell sc~nnlng spectrophotometer at 540 nm (such as an ELISA
plate reader), and over a range of cell densities a linear
relationship exists between formazan formation and cell number.
In the experiments measuring cell survival after UV, human cells
were seeded at 1,000 cells per well in a 96-well microtiter plate.
After overnight incubation, the media was removed and the cells
were irradiated with 254 nm UV at a fluence rate of 2.5 J/m /sec
for various doses of UV. Media and liposomes containing
endonuclease V were added and the cells were incubated for 5 days.
Fresh media containing 1 mg/ml MTT was added and the cells
incubated for 4 hours. The media was carefully removed leaving
the precipitated MTT formazan on the bottom of the wells, which
was solubilized by adding 50 ul per well of dimethyl sulfoxide and
incubating at 37C for 30 min. The plates were scanned by an
ELISA plate reader at 540 nm, and the optical densities of the UV
irradiated wells were compared to those of unirradiated wells to
determine cell survival.
(3) Results.
(A) Incision of UV-irradiated DNA. A typical result of
liposome-mediated incision of UV-irradiated DNA was as follows:
liposomes containing endonuclease V were prepared from a lipid
mixture of dipalmitoyl-L-alpha-phosphatidyl choline, phosphatidyl
choline and cholesterol (DPPC/PC/Chol; 7:2:1 molar ratio) at 20 mM
in an aqueous solution containing 0.2 mg/ml endonuclease V. The
liposome suspension had an optical density of 0.33 at 600 nm.
Confluent WI38 human fibroblast cells were irradiated with 100
J/m of UV and were treated with liposomes diluted 100-fold into
saline solution for 2 hrs. at 37C. The DNA was extracted and
single-stranded breaks were measured by alkaline gel
-38- l 3 3 8 6 4 3
electrophoresis. As an additional control, extracted DNA was
treated with purified endonuclease V to break the DNA at all sites
of pyrimidine dimers, and this DNA was also electrophoresed. The
gel was photographed, the negative scanned, and the number average
molecular weight and the breaks per million bases in DNA were
calculated.
The results of these experiments are shown in Table 2. As
shown therein, the UV treatment introduced (344.0 - 62.0) = 282
pyrimidine dimer sites per million bases in DNA, of which the
treatment with liposomes was able to incise (80.8 - 62.0) = 18.8
sites per million bases, or 6.7% of all sites. This frequency of
incision approaches the practical limit of 10-40% achieved with
mechanically disrupted WI38 cells and unencapsulated endonuclease
V as reviewed in the paper by Yarosh and Setlow, in MOLECULAR AND
CELLULAR BIOLOGY, 1981, referred to above.
(B) Repair replication. Typical results from experiments on
liposome-mediated repair replication were as follows: liposomes
were prepared as described in Example 4 and Table 1. Xeroderma
pigmentosum XP12BE cells and normal human epidermal keratinocytes
were equally divided and grown to near confluence in 60 mm dishes,
irradiated with 100 J/m2 of 254 nm W , and treated with various
concentrations of liposomes ("Endo V ug/ml" in Table 3). The cells
were pulse-labeled with [H-3]-thymidine for 4 hours, and the
amount of repair replication was determined by scintillation
counting, and expressed as a percentage of control values without
liposome treatment. The results of these experiments are shown in
Table 3.
The data in this table demonstrate that DNA repair synthesis
was increased up to 30% in normal human epidermal keratinocytes
treated with liposomes, in a manner proportional to the enzyme
concentration contributed by the liposomes. The PG/DCP liposomes
required about 10 times the enzyme concentration to achieve the
same biologic effect as PC/DCP liposomes. Similar results were
achieved with XP12BE cells, with a r~X; increase of 82%. The
larger effect in XP cells than in normal human cells is expected
_39_ 1 3 3 8 6 4 3
because in XP cells endo V restores DNA repair blocked by a
biochemical defect, whereas in normal human cells endo V augments
an already active process. As an additional control unirradiated
XP12BE cells were treated with PC/DCP liposomes, but no increase
in repair replication was observed.
(C) Cell survival after UV irradiation. Typical
results of two separate cell survival experiments using the
colorimetric assay were as follows: xeroderma pigmentosum XP12BE
cells were seeded in wells of 96-well plates at 1,000 cells per
well and incubated overnight. The media was removed and the cells
irradiated for various doses of UV. Media was added to the wells
along with liposomes containing endonuclease V. DPPC/PC/Chol and
PC/DCP liposomes were used at optical densities at 600 nm in the
media of 0.14 and 0.26, respectively. After 5 days incubation,
fresh media with MTT was added, and the plates were scanned after
4 hours incubation. The surviving fraction at each dose was
calculated by comparing the optical density of irradiated cells
with the optical density of unirradiated cells with the same
liposome treatment. The slope of the survival curve and the
correlation coefficient of the slope were calculated by linear
regression analysis of the log of the surviving fraction plotted
against UV dose. The results of these experiments are shown in
Figure 4 and in Table 4.
The theoretical basis for analysis of survival curves is
presented by Walter Harm in his book BIOLOGICAL EFFECTS OF
ULTRAVIOLET RADIATION, Cambridge University Press, Cambridge,
1980. As discussed in chapter 4 entitled "Inactivation of cells
and viruses" the fluence reduction factor is the ratio of the
slope of untreated cells to the slope of treated cells. It
represents the constant factor by which the biological effect of
UV irradiation has been attenuated, in this case the reduction in
lethality produced by liposome treatment. The fluence reduction
factors shown in Table 4 using liposomes approach the theoretical
limit of 2.29-2.89, when the gene for endonuclease V has been
inserted into xeroderma pigmentosum cells and the enzyme is
-40- 1 3 3 8 6 4 3
produced endogenously, as described by K. Valerie et al. in their
1987 CANCER RESEARCH paper referred to above.
No toxicity was observed in the unirradiated cells treated
with liposomes. Survival in these cells ranged between 87% and
123% compared to cells not treated with liposomes.
EXAMPLE 6
Enhancement of Human DNA Repair by
pH Sensitive Liposomes Cont~in;n~ Endonuclease V
(1) pH sensitive liposomes are superior to pH insensitive
liposomes in delivery of DNA repair enzymes to human cells.
(A) Liposomes composed of a mixture of phosphatidyl-
choline and phosphatidylethanolamine are more pH sensitive than
those composed of phosphatidylethanolamine alone. Liposomes were
prepared by the methods described in Example 3, using phospha-
tidylethanolamine, oleic acid and cholesteryl hemisuccinate
(PE/OA/CHEMS) in a 7:2:1 ratio or phosphatidylcholine, phospha-
tidylethanolamine, oleic acid and cholesteryl hemisuccinate
(PE/PC/OA/CHEMS) in a 3.5:3.5:2:1 ratio. Similar liposomes were
prepared substituting dipalmitoylphosphatidylcholine (DPPC) for
phosphatidylcholine and dipalmitoylphosphatidylethanolamine (DPPE)
for phosphatidylethanolamine.
The activity assay described in Example 3 was modified to
measure the pH sensitivity of liposomes. Liposomes were diluted
to 0.5 ug endonuclease V per ml into either 100 mM Tris pH 8, 200
mM NaCl, 2 mM EDTA, 2 mM DTT, or 100 mM citrate-phosphate pH 5,
200 mM NaCl, 2 mM EDTA, 2 mM DTT, or the pH 8 buffer containing 2%
Triton X-100. After incubation at 37C for 20 min, an equal
volume of plasmid substrate in water was added to each reaction,
and the incubation continued for 10 min. The samples were then
loaded on a 0.8% neutral agarose gel and the plasmid DNA analyzed
for breaks as described in Example 3. The results are shown in
Table 5. Liposomes with an equal mixture of phosphatidylethanol-
amine and phosphatidylcholine showed thirty times the release of
endonuclease V activity at pH 5 compared with liposomes containing
phosphatidylethanolamine alone. A difference of almost two-fold
-41- l 3 3 8 6 ~ 3
was found when the phospholipids were the synthetic DPPE and DPPC.
In each case the mixture of choline and ethanolamine head groups
in the lipid membrane produced greater pH sensitive destabiliza-
tion than the ethanolamine phospholipid alone. In addition, the
PE/PC mixture showed almost twice as much release of enzyme as did
the DPPE/DPPC mixture.
(B) Liposomes composed of 50% cholesteryl hemisuccinate are
more pH sensitive than liposomes composed of 10% cholesteryl hemi-
succinate. This assay for the pH sensitivity of liposomes is
based on the quenching of the fluorescent probe 8-aminonapthal-
ene-1,3,6-trisulfonic acid (ANTS) by a high concentration of
p-xylene-bis-pyridinium bromide (DPX) entrapped within the
liposomes, as described by Ben~z, Ellens and Szoka in their 1987
BIOCHEMISTRY paper cited above. Leakage from the liposomes
dilutes the DPX relative to the ANTS, the quenching is reduced and
fluorescence is increased.
Liposomes cont~;n;ng PC/PE/OA/CHEMS in ratios of either
3.5:3.5:2:1 or 2:2:1:5 and encapsulating 12.5 mM ANTS and 45 mM
DPX were diluted 1:400 into either 15 mM citrate-phosphate pH 5,
150 mM NaCl, 1 mM EDTA, or 20 mM Tris pH 8, 150 mM NaCl, 1 mM
EDTA, and incubated at 37C. As controls, liposomes were diluted
into identical buffers containing 1% Triton X-100 to dissolve the
liposomes, and incubated at 37C. Fluorescence was measured in a
Hoefer TK-100 (Trademark) fluorometer, with excitation peak at 365
nm and emission filter peak at 460 nm. Baseline was set as
fluorescence of liposomes at time zero, and 100% fluorescence was
set as the fluorescence in the Triton X-100 dissolved samples.
The results for the 2:2:1:5 liposomes are shown in Figure 5.
At pH 8, the fluorescence of the liposomes (closed circles) did
not change significantly during the incubation relative to the
fluorescence of the dissolved liposomes at pH 8 (set at 100%).
However, at pH 5 the fluorescence of the liposomes (open circles)
did increase relative to the dissolved liposomes (set at 100%)
during the 20 min. incubation. These results demonstrate that
these liposomes are destabilized by lowered pH, and release their
. , .
-42- 1 3 3 8 6 4 3
contents within 20 min. By contrast the 3.5:3.5:2:1 liposomes
showed much less pH destabilization, as shown in Table 6. At 20
minutes, when the 2:2:1:5 liposomes had released all their
contents, the 3.5:3.5:2:1 liposomes had released only 13% of
theirs, and after 3 hours only about one-third of the contents had
been released. The results demonstrate that the most pH sensitive
liposomes are composed of PC/PE/OA/CHEMS/ in a 2:2:1:5 molar
ratio.
(2) Biological assays for enhancement of DNA repair by pH
sensitive liposomes.
Liposomes were prepared from phosphatidylcholine, phospha-
tidylethanolamine, oleic acid and cholesteryl hemisuccinate in a
2:2:1:5 molar ratio, in the manner described in Example 3 using
the molecular sieve technique of Example 3 to isolate liposomes.
The activity of the enzyme entrapped in the liposomes was measured
by the activity assay described in Example 3 and the enzyme
concentration was measured by the ELISA method described in
Example 4. As a control, an aliquot of endonuclease V was boiled
for 60 minutes and liposomes were prepared as for the native
enzyme. The activity assay revealed no active enzyme in the
liposomes prepared from boiled endonuclease V. Human cells were
grown as described in Example 5, section (1), including the
SV40-transformed normal human fibroblast line GM637. Cells were
irradiated with the UV-C source described in Example 5.
(A) pH sensitive liposomes produced greater enhancement of
DNA repair than pH insensitive liposomes. Repair replication in
normal human epidermal keratinocytes after treatment with pH
sensitive liposomes was measured as described in Example 5, and
the results are shown in Table 7. pH sensitive liposomes achieved
almost ~x;r~l enhancement of DNA repair at 0.01 ug/ml. This may
be contrasted to the results with other liposomes as shown in
Table 3. No other liposome composition achieved this level of DNA
repair enhancement and no other liposome showed significant
biological activity at 0.01 ug/ml endonuclease concentration.
~43~ 1 3 3 8 6 4 3
The r- ~in;ng biological assays in this example used
PE/PC/OA/CHEMS liposomes at a molar ratio of 2:2:1:5.
(B) Unscheduled DNA synthesis (UDS) assay for enhanced DNA
repair. This assay is similar to the repair replication assay
described in Example 5, section 2B, and is also reviewed in the
1974 ANNALS OF INTERNAL MEDICINE paper by J. Robbins et al. In
the UDS assay, cells were grown on glass cover slides, irradiated
with UV, and then incubated with or without liposomes in media
containing 10 uCi/ml [H-3]-thymidine. After four hours the media
was replaced with fresh media containing 10 mM cold thymidine, and
incubated for an additional hour. The cells were then fixed with
acetone, and the coverslips coated with Kodak (Trademark) nuclear
track emulsion. After 7 days the coated coverslips were developed
with Kodak D-19 developer, and the cells examined microscopically.
Cells in S phase (replicating their entire DNA) during the four
hour incubation appeared to have dark black nuclei and were
excluded from analysis. Among cells not in S phase during the
incubation, the black grains over the nuclei of 25 randomly
selected cells were counted. The grains over these nuclei are
proportional to the amount of [H-3]-thymidine incorporated during
repair synthesis, and are a measure of DNA repair. This technique
has the advantage over the repair synthesis assay described in
Example 5 in that cells replicating their DNA during the repair
period are excluded from analysis, thus greatly reducing the
background against which DNA repair synthesis is measured.
The results of the UDS assay with W -irradiated normal human
keratinocytes incubated with media cont~;nlng different
concentrations of endonuclease V encapsulated in liposomes is
shown in Figure 6. Cells incubated without liposomes (open
circles) showed increased grains/nucleus with irradiation, as
would be expected for repair-proficient cells. However, treatment
with endonuclease V encapsulated in pH sensitive liposomes at 0.02
ug/ml (closed circles), 0.1 ug/ml (open squares) or 0.2 ug/ml
(closed squares) greatly enhanced their repair synthesis. Data
from this figure in addition to experiments with XP12BE cells from
,~
-44- l 3 3 8 6 4 3
a patient with xeroderma pigmentosum and with normal human GM637
cells are shown in Table 8. The results demonstrate that
treatment of UV-irradiated cells with endonuclease V enhanced
repair of DNA damage compared to irradiated control cells. The
increase was proportionately greater in XP compared to normal
cells, as was observed in the repair synthesis experiments
described in Example 5.
(C) Endonuclease-sensitive site (ESS) assay for enhanced
removal of DNA lesions. The ESS assay measures dimers in DNA as
sites sensitive to single-stranded breaks produced by T4
endonuclease V. DNA is purified from each sample and then either
treated or left untreated with T4 endonuclease V. The DNA is then
separated by size in fllk~l;ne agarose gel electrophoresis, and the
average molecular weight of the treated and untreated DNA is
measured as described in Example 5. Since the reciprocal of the
average molecular weight of the DNA represents the average number
of single-stranded breaks per unit DNA, the difference in the
average number of breaks between treated and untreated samples
represents the number of dimers per unit DNA, here expressed as
dimers per million DNA bases.
Human cells were irradiated and treated with either active or
inactive endo V encapsulated in liposomes at 0.3 ug enzyme/ml.
After 6 hours the DNA was extracted from each sample and the
number of pyrimidine dimers per million DNA bases was measured.
The results are shown in Table 9. For all cells, including normal
human epidermal keratinocytes and SV40-transformed fibroblasts
from a normal and an XP patient, treatment with the active
endonuclease in liposomes enhanced removal of dimers from DNA by
between 25 and 60%. The enhancement was greater in XP cells
because the liposomes restored repair which was blocked by the
biochemical defect, while in normal cells the liposomes augmented
an already active pathway.
(D) Colony forming ability assay for enhanced survival.
XP12BE cells from an XP patient were seeded at 500 and 5000 cells
per dish into tissue culture dishes and allowed to attach
~45~ l 3 3 8 6 4 3
overnight. The media was then removed from the dishes with 5000
cells and they were irradiated with 3 J/m2 of W-C. Fresh media
with 4% serum and with pH sensitive liposomes containing either
active or inactive endonuclease V, in addition to media controls
without liposomes, were added to all the cells, and they were
incubated overnight. The media was replaced with fresh media with
10% serum, and the cells incubated at 37C until they formed
colonies in lO days. The colonies were stained with Giemsa stain,
counted and the results are shown in Table 10. Treatment with
liposomes alone in the absence of UV reduced survival. However,
XP cells treated with liposomes containing active endonuclease V
survived the W irradiation at a much higher level than cells
treated with inactive endonuclease or no liposomes at all.
EXAMPLE 7
Enhancement of DNA Repair in Ani ~l.s
By Topical Skin Treatment with pH
Sensitive Liposomes Containing Endonculease V
(1) Treatment of mice with liposomes containing endonuclease
V in a topical cream. PC/PE/OA/CHEMS (2:2:1:5) pH sensitive
liposomes were prepared using active and inactive endonuclease V
by the methods described in Example 6. The liposomes were mixed
into a baby lotion (Johnson and Johnson, Skillman, New Jersey)
with 10% PBS to form a topical cream. Female mice of the SKH-l
hairless albino strain were obtained from Charles River Labs at 6
or 7 weeks of age. They were irradiated unrestrained with 10,000
J/m2 of W -B from two Westinghouse (Trademark) FS40 UV-B bulbs
whose fluence rate of 5-6 J/m2/sec was monitored with the UVX
radiometer using the W -B probe. The cream was applied to the
skin above the spine at 0.25 g/animal immediately after
irradiation. After six hours the ~n; ~1~ were sacrificed and a
strip of skin 5 x 20 mm above the spine was excised from each
animal and digested with 0.25% trypsin in PBS overnight at 4C.
The epidermis was scraped from the skin, and the DNA extracted and
purified. The dimer frequency in the purified DNA was measured by
the alkaline agarose gel electrophoresis assay described in
Examples 5 and 6.
,._ ` .~1 `1
. ,~;,
,~"r
- -46- 1 338643
(2) Results. The frequency of dimers in the epidermal DNA of
mice treated with pH sensitive liposomes is shown in Table 11. In
mice irradiated and untreated or treated with inactive
endonuclease V in liposomes, the dimer frequency in epidermal DNA
was between 90 and 96 per million bases. However, in mice treated
with liposomes containing active endonuclease V, the dimer
frequency was much lower, and the percent reduction in dimer
frequency was as great as 74%. This data demonstrates that
topical application of liposomes containing endonuclease V after
W exposure can penetrate the stratum corneum, enter epidermal
keratinocytes, and enhance removal of pyrimidine dimers in the DNA
of ~ n skin within six hours of irradiation.
Although specific embodiments of the invention have been
described and illustrated, it is to be understood that
modifications can be made without departing from the invention's
spirit and scope. For example, although the invention has been
illustrated in terms of DNA damage caused by ultraviolet light, it
is equally applicable to DNA damage resulting from other sources,
such as ionizing radiation, chemicals producing covalent adducts
to DNA, and other deformations of bases or strand breaks.
Similarly, in addition to being used after DNA damage has
occurred, as in the examples presented above, liposomes containing
DNA repair enzymes can be administered prophylactically prior to
the time cells will be exposed to conditions under which DNA
damage is likely.
1 338643
TABLE 1
Molar Optical Endo V Encapsulation
Liposome Ratio Density ug/ml Percentage
PC/DCP/Chol 7:2:1 7'5 5.2c 1.3
PC/stearylamine/Chol 7:2:1 4.8 4.9 1.2
DSPC/PC/Chol 7:2:10.12 0.2 0.3
DMPC/PC/Chol 7:2:10.23 0.2 0.16
DPPC/PC/Chol 7:2:118.0 1.2 0.24
PC/DCP 7:3 25.8 7.6 1.9
PG/DCP 7:3 5.0 38.0 9.5
PE/DCP 7:3 2.0 6.4 1.6
a Endo V concentration in dissolved liposomes measured using
standards of pure endo V. All measurements were in
duplicate.
b Percentage of initial enzyme entrapped in liposome
preparation.
c Concentration = 1,500 units/ml.
d Concentration = 6,600 units/ml.
TABLE 2
Number Average Breaks per
Treatment Mol Wt (bases) million bases
none 16,117 62.0
DPPC/PC/Chol 12,381 80.8
DPPC/PC/Chol + Endo-
nuclease V 2,906 344.0
-
-48-
1 338643
TABLE 3
PERCENT
CELL J/m2 LIPOSOMEENDO V REPAIR
~ REPLICATION
Normal Human100 PC/DCP 0 100
Epidermal 0.075 106
Keratinocytes 0.15 123
0.375 125
100 PG/DCP 0 100
0.75 106
1.5 110
3.0 130
100 DPPC/PC/Chol 0 100
0.05 121
0.1 130
0.25 113
XP12BE 100 PC/DCP 0 100
0.075 117
0.15 144
0.375 182
0 PC/DCP 0 100
0.075 100
0.15 96
0.375 95
100 PC/DCP/Chol 0 100
0.04 116
0.08 117
100 DPPC/PC/Chol 0 100
0.05 157
0.1 105
0.25 130
100 PG/DCP 0 100
0.75 104
1.5 106
3.0 118
100 PE/DCP O 100
0.1 122
0.25 122
0.5 144
-49-
1 338643
TABLE 4
Fluence
Survival Correlation Reduction
Liposome Treatment Slope Coefficient Factor
none -0.061 0.95
2.05
DPPC/PC/Chol -0.030 0.96
none -0.043 0.99
1.57
PC/DCP -0.027 0.95
TABLE 5
Excess W -specific breaks
Liposome Compositionper plasmid: pH 5 over pH 8
PE/OA/CHEMS 7:2:1 0.01
PE/PC/OA/CHEMS 3.5:3.5:2:1 0.29
DPPE/OA/CHEMS 7:2:1 0.09
DPPE/DPPC/OA/CHEMS 3.5:3.5:2:1 0.15
- 1 338643
-50-
TABLE 6
Percent Fluorescence at pH 5
PC/PE/OA/CHEMS ratio
Minutes 3.5:3.5:2:1 2:2:1:5
O 0% 0%
3 - 54
2 81
13 100
14
120 23
180
TABLE 7
PE/PC/OA/CHEMS Liposomes (2:2:1:5)
Endo V ug/mlPercent control repair replication
none 100%
0.01 142
0.10 166
0.25 165
0.50 169
l.O0 156
1 338643
-51-
TABLE 8
UDS Percent Control
Endo V in Liposomes J/m2 NHK XP12BE GM637
0.20 ug/ml 10 340 564 142
343 679 150
0.10 ug/ml 10 251 297 96
234 374 92
0.05 ug/ml 10 99
71
0.02 ug/ml 10 164 135
146 242
Control was irradiated cells untreated with liposomes.
Control was irradiated cells treated with liposomes
containing inactive endonuclease V.
TABLE 9
Dimers Per Million DNA Bases
Liposome Treatment
Cell LineActive Inactive % Reduction
Normal human 88.0 119.7 27%
keratinocytes
GM637 - human 94.0 136.2 31%
fibroblasts
XP12BE - XP 43.9 106.8 59%
fibroblasts
-
-52- 1 3 3 8 6 4 3
TABLE 10
Percent Survival of XP12BE
Endo V in Liposomes
(ug/ml) without UV with 25 J/m2% Control
none 100% 1.3% 100%
Inactive -- 1.5 83 0.9 68
-- 0.15 53 1.5 115
-- 0.015 55 1.8 138
Active -- 1.5 20 3.6 277
-- 0.15 42 4.9 382
-- 0.015 43 2.5 192
TABLE 11
Mouse Skin
Endo V in Liposomes Dimers Per Million Bases % Reduction
none 95.3
2.0 ug/ml - active72.9 22%
- inactive 93.0
0.5 ug/ml - active23.8 74%
- inactive 90.8
0.1 ug/ml - active55.9 40%
- inactive 92.9
