Method and composition for regulating apoptosis

Methods and compositions for preventing or inhibiting apoptosis are provided by this invention. The methods require introducing into a cell which may undergo apoptosis a nucleic acid molecule coding for a gene product having crmA biological activity or a crmA polypeptide. This invention also provides compositions and methods for maintaining T cell viability in a subject infected with the human immunodeficiency virus (HIV), by administering to the subject an effective amount of a nucleic acid molecule coding for a gene product having crmA biological activity or the gene product itself.

FIELD OF THE INVENTION 
This invention relates to methods for regulating apoptosis in a population 
of cells as well as compositions useful to regulate apoptosis. 
BACKGROUND OF THE INVENTION 
Apoptosis, or programmed cell death (PCD) is a type of cell death that is 
fundamentally distinct from degenerative death or necrosis. It is an 
active process of gene-directed cellular self-destruction which in some 
instances, serves a biologically meaningful homeostatic function. This can 
be contrasted to necrosis which is cell death occurring as the result of 
severe injurious changes in the environment of infected cells. For a 
general review of apoptosis, see Tomei, L. D. and Cope, F. O. Asoptosis: 
The Molecular Basis of Cell Death (1991) Cold Spring Harbor Press, N.Y.; 
Tomei, L. D. and Cope, F. O. Apoptosis II: The Molecular Basis of 
Apoptosis in Disease (1994) Cold Spring Harbor Press, N.Y.; and Duvall and 
Wyllie (1986) Immun. Today 7(4):115-119. 
Morphologically, apoptosis is characterized by the rapid condensation of 
the cell with preservation of membranes. Synchronistically with the 
compaction of chromatin, several biochemical changes occur in the cell. 
Nuclear DNA is cleaved at the linker regions between nucleosomes to 
produce fragments which are easily demonstrated by agarose gel 
electrophoresis wherein a characteristic ladder develops. 
Apoptosis has been linked to many biological processes, including 
embryogenesis, development of the immune system, elimination of 
virus-infected cells, and the maintenance of tissue homeostasis. Apoptosis 
also occurs as a result of human immunodeficiency virus (HIV) infection of 
CD4.sup.+ T lymphocytes (T cells). Indeed, one of the major 
characteristics of AIDS is the gradual depletion of CD4.sup.+ T 
lymphocytes during the development of the disease. Several mechanisms, 
including apoptosis, have been suggested to be responsible for the CD4 
depletion. It is speculated that apoptotic mechanisms might be mediated 
either directly or by the virus replication as a consequence of the HIV 
envelope gene expression, or indirectly by priming uninfected cells to 
apoptosis when triggered by different agents. 
The depletion of CD4.sup.+ T cells results in the impairment of the 
cellular immune response. It has been proposed that an inappropriate 
activation-induced T cell PCD causes the functional and numerical 
abnormalities of T.sub.H cells from HIV-infected patients, that leads to 
the near collapse of the patient's immune system. 
Therefore, it is advantageous to block apoptosis and the ensuing depletion 
of T cells. Accordingly, a need exists to maintain T cell function and 
viability in HIV infected individuals and to provide systems to screen for 
new drugs that may assist in maintaining the cellular immune response. 
This invention satisfies this need and provides related advantages as 
well. 
SUMMARY OF THE INVENTION 
This invention provides compositions and methods for preventing or 
inhibiting apoptosis in a suitable cell by introducing into the cell a 
nucleic acid molecule coding for a gene product having crmA biological 
activity or alternatively, the crmA gene product. 
Also provided by this invention are compositions and methods for preventing 
or inhibiting induced apoptosis in a suitable cell by introducing into the 
cell a nucleic acid molecule coding for a gene product having cytokine 
response modifier crmA biological activity or the gene product so that 
induced apoptosis is prevented or inhibited. 
Further provided by this invention are compositions and methods for 
maintaining T cell viability in a subject infected with or susceptible to 
infection with the human immunodeficiency virus by administering to the 
subject an effective amount of a nucleic acid molecule coding for a gene 
product having crmA biological activity or the crmA gene product.

DETAILED DESCRIPTION OF THE INVENTION 
As is known to those of skill in the art, apoptosis is an active process of 
gene-directed cellular self-destruction. This invention provides 
compositions and methods for preventing or inhibiting apoptosis in a 
suitable cell or a population of suitable cells by introducing into the 
cell or cells an effective amount of a nucleic acid molecule coding for a 
gene product having crmA biological activity. The method also may be 
practiced using the gene product itself. It is important to note that the 
method of this invention inhibits apoptosis even in the presence of 
apoptotic-inducing agents, such as receptor ligands, e.g., anti-TCR, tumor 
necrosis factor (TNF), HIV, SIV or anti-Fas antibody. Accordingly, this 
method provides an improvement over prior art methods wherein apoptosis 
can be inhibited by interfering with the induction pathway at the level of 
ligand induction, such as by providing antibodies or anti-ligand 
antibodies to interfere with the binding of the ligand to its cell surface 
receptor. However, this invention can be combined with the use of such 
prior art methods to inhibit apoptosis. 
The terms "preventing" or "inhibiting" are intended to mean a reduction in 
cell death or a prolongation in the survival time of the cell. They also 
are intended to mean a diminution in the appearance or a delay in the 
appearance of morphological and/or biochemical changes normally associated 
with apoptosis. Thus, this invention provides compositions and methods to 
increase survival time and/or survival rate of a cell or population of 
cells which, absent the use of the method, would normally be expected to 
die. Accordingly, it also provides compositions and methods to prevent or 
treat diseases or pathological conditions associated with unwanted cell 
death in a subject. 
Suitable cells or "target cells" for the practice of this method include, 
but are not limited to, cells that are induced to PCD by an endogenous 
agent such as HIV, anti-TCR antibody, TNF and anti-Fas antibody. In one 
embodiment, these cells constitutively and inducibly express receptors for 
either or both of the cytokine tumor necrosis factor (TNF) or the cell 
death transducing receptor Fas or TCR and which have been activated by 
their respective ligand. Recently, three separate groups have reported 
that Fas-induced apoptosis is involved in T cell death. Specifically, one 
group has shown that the Fas receptor, which can transduce a potent 
apoptotic signal when ligated, is rapidly expressed following activation 
on T cell hybridomas. It was suggested that the Fas receptor-ligand 
interaction induces cell death in a cell-autonomous manner. See Dhein et 
al. (1995) Nature 373:438-441; Brunner et al. (1995) Nature 373:441-444; 
and Ju et al. (1995) Nature 373:444-448. 
For the purpose of illustration only, examples of suitable cells are T 
lymphocytes (T cells) (e.g., TCR.sup.+, CD4.sup.+ and CD8.sup.+ T cells) 
leukocytes and mixed leukocyte cultures (MLC), B lymphoma cells (e.g., 
A202J (ATCC)), bone marrow cells, endothelial cells, breast carcinoma 
cells, fibroblast cells, epithelial tumor cells (see Spriggs, D. R. et al. 
(1988) J. Clin. Inves. 81:455-460) and monocytes. Fas and TNF receptor 
expression also has been identified on numerous tissues, see for example 
Watanabe-Fukunaga et al. (1992) J. Immun. 148:1274-1279 and Owen-Schaub, 
L. B. et al. (1994) Cancer Res. 54:1580-1586; Dhein et al. (1995) Nature 
373:438-441; Brunner et al. (1995) Nature 373:441-444; and Ju et al. 
(1995) Nature 373:444-448. Assays for identifying additional "suitable" 
cells sensitive to induction or activation, e.g., TCR-, TNF- or 
Fas-related apoptosis, are well known to those of skill in the art. (See 
for example, Opipari, et al. J. Biol. Chem. (1992) 267:12424-12427; 
Yonehara et al. J. Exp. Med. (1989) 169:1747-1756; Dhein et al. (1995) 
supra; Brunner et al. (1995) supra and Ju et al. (1995) supra) However, 
this method is particularly suitable for use with TCR.sup.+, CD8.sup.+ or 
CD4.sup.+ T cells or tissues that harbor the simian immunodeficiency 
virus (SIV) or alternatively, the human immunodeficiency virus (HIV). The 
cells can be mammalian cells or animal cells, such as guinea pig cells, 
rabbit cells, simian cells, mouse cells, rat cells, or human cells. They 
can be continuously cultured or isolated from an animal or human. In a 
separate embodiment of this invention, neurological cells are specifically 
excluded. 
This invention is based on Applicants' finding that the cowpox virus crmA 
gene product is an exceptionally potent inhibitor of apoptosis induced by 
binding of a cell surface receptor to its ligand, e.g., TCR ligand, HIV, 
Fas or TNF. In one embodiment which utilized TNF- and Fas- pathways; it is 
capable of blocking the cell death program even at pharmacological doses 
of the death stimulus. crmA is a cowpox virus gene which encodes a 
protease inhibitor of the serpin family. The nucleic acid and 
corresponding amino acid sequences of crmA have been reported (Pickup et 
al., Proc. Natl. Acad. Sci. (1986) 83:7698-7702) and are shown in FIG. 5. 
The only reported target for the crmA protein is the cysteine protease 
interleukin-1.beta. converting enzyme (ICE). However, Applicants have 
found that crmA is an exceptionally potent inhibitor of apoptosis. 
Therefore, an important new function for crmA is the prevention or 
inhibition of ligand-induced or cytokine-induced apoptosis. Further, the 
data suggest that a protease, either ICE or a related crmA-inhibitable 
protein, is a component of the Fas- and TNF-induced cell death pathways. 
Thus, this invention provides: compositions and methods for preventing or 
inhibiting ligand-induced or cytokine-induced apoptosis; an assay for 
determining drugs or agents which facilitate or prevent or inhibit 
apoptosis; an assay for drugs to treat or ameliorate the symptoms 
associated with a disease or pathological conditions that occur as a 
result of apoptosis (such as AIDS); an assay for detecting the protease 
involved in the Fas- and TNF- induced cell death pathways, as well the 
proteases discovered using this method. 
The crmA gene or nucleic acid can be isolated from natural or native 
sources as described in Pickup et al. (1986) supra. The term "native" 
refers to the form of a nucleic acid, protein, polypeptide, antibody or a 
fragment thereof that is isolated from nature or which is without an 
intentional amino acid substitution. As used herein, "nucleic acid" and 
"gene" are synonymous and shall mean single and double stranded genomic 
DNA, cDNA, mRNA and cRNA. "Isolated" when used to describe the state of 
the nucleic acids or proteins, denotes the nucleic acids or proteins free 
of at least a portion of the molecules associated with or occurring with 
nucleic acids in their native environment. 
Reference is made to standard textbooks of molecular biology that contain 
definitions and methods and means for carrying out basic techniques, 
encompassed by the present invention. See, for example, Maniatis et al., 
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New 
York (1982) and Sambrook et al., Molecular Cloning: A Laboratory Manual, 
Cold Spring Harbor Laboratory, New York (1989) and the various references 
cited therein. This reference and the cited publications are expressly 
incorporated by reference into this specification. 
The DNA sequence provided in FIG. 5 can be duplicated using a DNA sequencer 
and methods well known to those of skill in the art. For example, the 
sequence can be chemically replicated using PCR (Perkin-Elmer) which in 
combination with the synthesis of oligonucleotides, allows easy 
reproduction of DNA sequences. A DNA segment of up to approximately 6000 
base pairs in length can be amplified exponentially starting from as 
little as a single gene copy by means of PCR. In this technique, a 
denatured DNA sample is incubated with two oligonucleotide primers that 
direct the DNA polymerase-dependent synthesis of new complementary 
strands. Multiple cycles of synthesis each afford an approximate doubling 
of the amount of target sequence. Each cycle is controlled by varying the 
temperature to permit denaturation of the DNA strands, annealing the 
primers, and synthesizing new DNA strands. The use of a thermostable DNA 
polymerase eliminates the necessity of adding new enzyme for each cycle, 
thus permitting fully automated DNA amplification. Twenty-five 
amplification cycles increase the amount of target sequence by 
approximately 10.sup.6 -fold. The PCR technology is the subject matter of 
U.S. Pat. Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202. 
The nucleic acid can be duplicated using a host-vector system and 
traditional cloning techniques with appropriate replication vectors. A 
"host-vector system" refers to host cells which have been transfected with 
appropriate vectors using recombinant DNA techniques. The vectors and 
methods disclosed herein are suitable for use in host cells over a wide 
range of eucaryotic organisms. This invention also encompasses the cells 
transformed with the novel replication and expression vectors described 
herein. 
Indeed, the crmA gene can be duplicated in many replication vectors such as 
the vaccinia virus as described in Pickup et al. (1986) supra, and 
isolated using methods described in Sambrook et al. (1989) supra. 
The crmA gene made and isolated using the above methods can be directly 
inserted into an expression vector, such pcDNA3 (Invitrogen) and inserted 
into a suitable animal or mammalian cell such as a guinea pig cell, a 
rabbit cell, a simian cell, a mouse, a rat or a human cell. 
In the practice of one embodiment of this invention, the crmA nucleic acid 
molecule is introduced into the cell and expressed and cell death is 
aborted. A variety of different gene transfer approaches are available to 
deliver the crmA gene into a target cell, cells or tissues. Among these 
are several non-viral vectors, including DNA/liposome complexes, and 
targeted viral protein DNA complexes. To enhance delivery to a cell, the 
nucleic acid or proteins of this invention can be conjugated to antibodies 
or binding fragments thereof which bind cell surface antigens, e.g., TCR, 
CD3 or CD4. Liposomes that also comprise a targeting antibody or fragment 
thereof can be used in the methods of this invention. This invention also 
provides the targeting complexes for use in the methods disclosed herein. 
The crmA nucleic acid also can be incorporated into a "heterologous DNA" or 
"expression vector" for the practice of this invention. The term 
"heterologous DNA" is intended to encompass a DNA polymer such as viral 
vector DNA, plasmid vector DNA or cosmid vector DNA. Prior to insertion 
into the vector, it is in the form of a separate fragment, or as a 
component of a larger DNA construct, which has been derived from DNA 
isolated at least once in substantially pure form, i.e., free of 
contaminating endogenous materials and in a quantity or concentration 
enabling identification, manipulation, and recovery of the segment and its 
component nucleotide sequences by standard biochemical methods, for 
example, using a cloning vector. As used herein, "recombinant" is intended 
to mean that a particular DNA sequence is the product of various 
combination of cloning, restriction, and ligation steps resulting in a 
construct having a sequence distinguishable from homologous sequences 
found in natural systems. Recombinant sequences can be assembled from 
cloned fragments and short oligonucleotides linkers, or from a series of 
oligonucleotides. 
As noted above, one means to introduce the nucleic acid into the cell of 
interest is by the use of a recombinant expression vector. "Recombinant 
expression vector" includes vectors which are capable of expressing DNA 
sequences contained therein, where such sequences are operatively linked 
to other sequences capable of effecting their expression. It is implied, 
although not always explicitly stated, that these expression vectors must 
be replicable in the host organisms either as episomes or as an integral 
part of the chromosomal DNA. In sum, "expression vector" is given a 
functional definition, and any DNA sequence which is capable of effecting 
expression of a specified DNA sequence disposed therein is included in 
this term as it is applied to the specified sequence. Suitable expression 
vectors include viral vectors, including adenoviruses, adeno-associated 
viruses, retroviruses, cosmids and others. Adenoviral vectors are a 
particularly effective means for introducing genes into tissues in vivo 
because of their high level of expression and efficient transformation of 
cells both in vitro and in vivo. 
Replication-incompetent retroviral vectors also can be used with this 
invention. As used herein, the term "retroviral" includes, but is not 
limited to, a vector or delivery vehicle having the ability to selectively 
target and introduce the coding sequence into dividing cells, such as the 
mouse molony-leukemia virus. As used herein, the terms 
"replication-incompetent" is defined as the inability to produce viral 
proteins, precluding spread of the vector in the infected host cell. As 
should be understood by those of skill in the art, crmA nucleic acid will 
be ribonucleic acid (RNA) for introduction with a retroviral vector. 
Another example of a replication-incompetent retroviral vector is LNL6 
(Miller, A. D. et al., BioTechnicues 7:980-990 (1989)). The methodology of 
using replication-incompetent retroviruses for retroviral-mediated gene 
transfer of gene markers is well established (Correll, P. H. et al., 
(1989) PNAS USA 86:8912; Bordignon, C. et al., PNAS USA (1989) 86:6748-52; 
Culver, K. et al., (1991) PNAS USA 88:3155; Rill, D. R. et al., (1990) 
Blood 79(10):2694-700). Clinical investigations have shown that there are 
few or no adverse effects associated with these viral vectors (Anderson, 
(1992) Science 256:808-13). 
In one embodiment of this invention, the expression vector is to be 
specifically targeted to T cells. For these methods, it intended that the 
crmA DNA be operatively linked to a promoter that is highly active in T 
cells. Such promoters include, but are not limited to: IFN-.tau.; IL-2; 
IL-3; IL-4; IL-5; IL-9; IL-10; TFN-.beta.; GM-CSF; CD4, CD8 and the IL-2 
promoter. 
Although the method is preferably practiced with the crmA gene, it should 
be apparent to those of skill in the art that the polypeptide product of 
the crmA gene and its biological equivalents are useful in the methods of 
this invention. The crmA gene product is a 38 kDa protein and is known to 
be a specific inhibitor of IL-1.beta.. It can be purified from natural 
sources as described in Ray, C. A. (1992) Cell 69:597-604 or produced 
recombinantly using the expression vectors described above in a 
host-vector system such as described in Pickup et al. (1986) supra, Ray et 
al. (1992) supra and Moss, B. ed. (1990) Virology, pp:2079-2112, Raven 
Press, N.Y. The protein is used in substantially pure form. By 
"substantially pure," it is meant that the protein is substantially free 
of other biochemical moieties with which it is normally associated in 
nature. The proteins also can be produced using the sequence provided in 
FIG. 5 and methods well known to those of skill in the art. 
Accordingly, this invention also provides a crmA polypeptide, protein, a 
biological equivalent thereof and fusion proteins containing these, for 
use in the methods described herein. The polypeptides or proteins can be 
conjugated to targeting antibodies, such as anti-CD3 or anti-CD4 for 
targeted delivery to T cells. 
A "biological equivalent" is intended to mean any fragment of the nucleic 
acid or protein, a mimetic (protein and non-protein mimetic) also having 
the ability to inhibit apoptosis using the assay system described and 
exemplified herein. For example, purified crmA polypeptide can be 
contacted with a suitable cell as described above and under such 
conditions that apoptosis is inhibited. It is understood that limited 
modifications can be made to the primary sequence of the crmA sequence as 
shown in FIG. 5 and used in this invention without destroying its 
biological function, and that only a portion of the entire primary 
structure may be required in order to effect biological activity. Examples 
of such biological equivalent fragments include, but are not limited to 
the 5.2 kb EcoRI fragment or the 2.7 kb BglII fragment and the 
polypeptides encoded by these nucleic acid molecules as described in 
Pickup et al. (1986) supra. It is further understood that minor 
modifications of the primary amino acid sequence may result in proteins 
which have substantially equivalent or enhanced function as compared to 
the molecule within the vector. These modifications may be deliberate, as 
through site-directed mutagenesis, or may be accidental such as through 
mutation in hosts. All of these modifications are included as long as the 
ability to inhibit apoptosis is retained. 
This method can be practiced in vitro, ex vivo or in vivo. When the method 
is practiced in vitro, the expression vector, protein or polypeptide can 
be added to the cells in culture or added to a pharmaceutically acceptable 
carrier as defined below. In addition, the expression vector or crmA DNA 
can be inserted into the target cell using well known techniques such as 
transfection, electroporation or microinjection. 
More specifically, the in vitro method comprises providing cell cultures or 
tissue cultures having either a cell surface receptor that mediates 
apoptosis such as a TCR, the TNF receptor or the Fas receptor. The cells 
are cultured under conditions (temperature, growth or culture medium and 
gas (CO.sub.2)) and for an appropriate amount of time to attain 
exponential proliferation without density dependent constraints. The cells 
are then exposed to preliminary conditions necessary for apoptosis, for 
example an effective amount of an inducing agent, e.g., a TCR ligand, HIV, 
SIV, TNF, or a Fas ligand such as an anti-Fas antibody is added to the 
culture. Anti-Fas antibodies and mitogens (ConA) are well known to those 
of skill in the art. (Itoh, N. et al. (1991) Cell 66:233-243 and Yonehara 
et al. (1989) J. Exp. Med. (1989) 169:1747-1756). These cells are now 
"induced" to apoptosis. The cells are again cultured under suitable 
temperature and time conditions. In one embodiment, HIV or SIV is added to 
the culture. In other embodiments, a drug or agent to be tested is added 
in varying concentrations at a time that is simultaneous with, prior to, 
or after the inducing agent. 
The crmA nucleic acid or protein is then added to the culture in an 
effective amount and the cells are cultured under suitable temperature and 
time conditions to inhibit apoptosis. The crmA nucleic acid or protein can 
be added prior to, simultaneously with, or after, the inducing agent. The 
cells are assayed for apoptotic activity using methods well known to those 
of skill in the art and described herein. It is apparent to those of skill 
in the art that two separate culture of cells must be treated and 
maintained as the test population. One is maintained without receiving an 
inducing agent to determine background release and the second without 
receiving the agent to be tested. The second population of cells acts as a 
control. 
The use of the compositions and methods in vitro provides a powerful 
bioassay for screening for drugs which are agonists or antagonists of crmA 
function in these cells. Thus, one can screen for drugs having similar or 
enhanced ability to prevent or inhibit apoptosis. It also is useful to 
assay for drugs having the ability to inhibit HIV infection and 
replication, since the CD4.sup.+ cell will not die as a result of the 
concurrent viral infection. One of skill in the art can determine when the 
method has been successfully performed by noting the absence of apoptotic 
morphological changes or more simply, by the absence of cell death. The in 
vitro method further provides an assay to determine if the method of this 
invention is useful to treat a subject's pathological condition or disease 
that has been linked to apoptotic cell death in the individual. 
For example, a T cell hybridoma cell line such as Jurkat can be stably 
transfected with the crmA expression construct or vector alone and clonal 
cell lines derived. Transfection of Jurkat cell by electroporation can be 
performed as described in Laherty et al. J. Biol. Chem. (1993) 
263:5032-5039. crmA-expressing and vector-transfected control cells are 
.sup.51 Cr-labeled and plated (5.times.10.sup.5 /ml) on untreated or 
anti-CD3 (available from the cell line 145-2C11 (ATCC)) treated tissue 
culture plastic plates. Cells cultured on uncoated cells are used to 
determine background release. The percentage cell death will be determined 
at various times after culture by the formula: c.p.m. released from the 
experimental group minus c.p.m. of background release divided by c.p.m. 
released by 0.5% Triton X-100 (complete lysis)--c.p.m. of background 
release. 
A substantial decrease in percent cell death induced by plating cells on 
immobilized anti-CD3 monoclonal antibody is an indication that crmA 
inhibits T cell receptor-induced death. Using the method described above, 
various agents can be tested for their ability to inhibit or prevent 
apoptosis. 
In a separate embodiment, the T cell line designated CEM (ATCC) is obtained 
and used because it has been shown to undergo PCD upon infection with HIV. 
CEM cells are transfected by electroporation with the crmA expression 
construct and vector alone as control. Clonal lines are derived and 
infected at various multiplicity of infection ratios with HIV. Cytopathic 
effect is assayed by microscopic observation and apoptosis quantitated 
following propidium iodine staining. Using the method described above, 
various agents can be tested for their ability to inhibit or prevent 
apoptosis. 
When the method is practiced in vivo in a human patient, it is unnecessary 
to provide the inducing agent since it is provided by the patient's immune 
system. However, when practiced in an experimental animal model, it can be 
necessary to provide an effective amount of the inducing agent in a 
pharmaceutically acceptable carrier prior to administration of the crmA 
product, to induce apoptosis. When the method is practiced in vivo, the 
carrying vector, crmA polypeptide, polypeptide equivalent, or crmA 
expression vector can be added to a pharmaceutically acceptable carrier 
and systemically administered to the subject, such as a human patient or 
an animal such as a mouse, a guinea pig, a simian, a rabbit or a rat. 
Alternatively, it can be directly infused into the cell by microinjection. 
A fusion protein also can be constructed comprising the 
protease-inhibitory region of the crmA, diphtheria toxin and a T-cell 
specific ligand for targeting to a T cell. Such T cell specific ligands 
include, but are not limited to anti-CD3, anti-CD4, anti-CD28 and 
anti-IL-1 antibody protein. 
Acceptable "pharmaceutical carriers" are well known to those of skill in 
the art and can include, but not be limited to any of the standard 
pharmaceutical carriers, such as phosphate buffered saline, water and 
emulsions, such as oil/water emulsions and various types of wetting 
agents. 
When practiced in vivo, the compositions and methods are particularly 
useful for maintaining T cell viability and function in a subject or an 
individual suffering from or predisposed to suffer from abnormal 
lymphocyte death. When the animal is an experimental animal such as a 
simian (using SIV), this method provides a powerful assay to screen for 
new drugs that may be used alone or in combination with this invention to 
ameliorate or reduce the symptoms and opportunistic infections associated 
with HIV infection or AIDS. 
This invention also is particularly useful to ward off lymphocyte death or 
immunosuppression in AIDS patients. By preventing or inhibiting apoptosis, 
not only is cell death prevented but functionality, e.g., 
immuno-proliferative capacity, is restored to the cell and a responsive 
immune system is retained or regained. Accordingly, the compositions and 
methods of this invention are suitably combined with compositions and 
methods which prevent or inhibit HIV infectivity and replication. 
The method can also be practiced ex vivo using a modification of the method 
described in Lum et al. (1993) Bone Marrow Transplantation 12:565-571. 
Generally, a sample of cells such as bone marrow cells or MLC can be 
removed from a subject or animal using methods well known to those of 
skill in the art. An effective amount of crmA nucleic acid is added to the 
cells and the cells are cultured under conditions that favor 
internalization of the nucleic acid by the cells. The transformed cells 
are then returned or reintroduced to the same subject or animal 
(autologous) or one of the same species (allogeneic) in an effective 
amount and in combination with appropriate pharmaceutical compositions and 
carriers. 
As used herein, the term "administering" for in vivo and ex vivo purposes 
means providing the subject with an effective amount of the nucleic acid 
molecule or polypeptide effective to prevent or inhibit apoptosis of the 
target cell. Methods of administering pharmaceutical compositions are well 
known to those of skill in the art and include, but are not limited to, 
microinjection, intravenous or parenteral administration. The compositions 
are intended for topical, oral, or local administration as well as 
intravenously, subcutaneously, or intramuscularly. Administration can be 
effected continuously or intermittently throughout the course of 
treatment. Methods of determining the most effective means and dosage of 
administration are well known to those of skill in the art and will vary 
with the vector used for therapy, the polypeptide or protein used for 
therapy, the purpose of the therapy, the target cell being treated, and 
the subject being treated. Single or multiple administrations can be 
carried out with the dose level and pattern being selected by the treating 
physician. For example, the compositions can be administered prior to a 
subject already suffering from a disease or condition that is linked to 
apoptosis. In this situation, an effective "therapeutic amount" of the 
composition is administered to prevent or at least partially arrest 
apoptosis and the accompanying pathology such as immunosuppression in HIV 
infected individuals. 
However, the compositions can be administered to subjects or individuals 
susceptible to or at risk of developing apoptosis-related disease to 
prevent pathological cell death. In one embodiment, the composition can be 
administered to a subject susceptible to HIV-related lymphocyte 
disfunction to maintain lymphocyte cell function and viability. In these 
methods a "prophylactically effective amount" of the composition is 
administered to maintain cellular viability and function at a level near 
to the pre-infection level. 
It should be understood that by preventing or inhibiting unwanted cell 
death in a subject or individual, the compositions and methods of this 
invention also provide methods for treating, preventing or ameliorating 
the symptoms associated with a disease characterized by apoptosis of 
cells. Such diseases include but are not limited to AIDS, acute and 
chronic inflammatory disease, leukemia, myocardial infarction, stroke, 
traumatic brain injury, neural and muscular degenerative diseases, aging, 
tumor induced-cachexia and hair loss. 
This invention also provides vector and protein compositions useful for the 
preparation of medicaments which can be used for preventing or inhibiting 
apoptosis, maintaining cellular function and viability in a suitable cell 
or for the treatment of a disease characterized by the unwanted death of 
target cells. 
It is to be understood that while the invention has been described in 
conjunction with the above embodiments, that the foregoing description and 
the following examples are intended to illustrate and not limit the scope 
of the invention. Other aspects, advantages and modifications within the 
scope of the invention will be apparent to those skilled in the art to 
which the invention pertains. 
Experiment I 
Analysis of Apoptosis--Apoptosis was assessed by the use of fluorescent 
DNA-staining dyes to reveal nuclear morphology and by transmission 
electron microscopy. For propidium iodide staining, MCF7 cells were grown 
on 22 mm.sup.2 No. 1 glass coverslips (Corning) placed in 35 mm wells of a 
6-well culture dish (Costar). Following treatment with TNF, anti-Fas 
cycloheximide (CHX), or no treatment, medium was removed and the wells 
were rinsed twice with phosphate buffered saline (PBS), fixed in 100% 
methanol at -20.degree. C. for 10 minutes, washed three times with PBS, 
and stained at room temperature for 10 minutes in a 100 .mu.g/ml solution 
or propidium iodide (Sigma) made in PBS. The coverslips were then washed 
three times with PBS, blotted dry and mounted onto glass slides using 
Vectashield mounting medium for fluorescence (Vector Laboratories). BJAB 
cells were stained using acridine orange (SIGMA) by preparing a wet mount 
of 30 .mu.l of a cell suspension at a density of approximately 
3.times.10.sup.5 cell/ml mixed with 5 .mu.l of a 100 .mu.g/ml acridine 
orange solution made in PBS. Both propidium iodide-stained MCF7 and 
acridine orange-stained BJAB nuclei were visualized by fluorescence 
microscopy using a FITC range barrier filter cube. Laser-scanning confocal 
microscopy was performed using the Bio-Rad MRC 600 confocal microscope and 
digitized images obtained were artificially colorized. 
For electron microscopy, cells were fixed and processed as per standard 
electron microscopy procedures. 
Experiment II 
Quantitative Apoptosis Assays--MCF7 cells or derived transfectants were 
plated at a concentration of 2.5.times.10.sup.5 cells/well onto glass 
coverslips. Two days later, after the cells had adhered and spread, TNF or 
anti-Fas+CHK were added. TNF was added at a final concentration of 20 
ng/ml, anti-Fas at 25 ng/ml, and CHX (Sigma) at 10 .mu.g/ml. After 22 
hours for the TNF treated samples or after 18 hours for the anti-Fas+CHX 
treated samples, cells were fixed, stained with propidium iodide and 
mounted as described above. Apoptotic and non-apoptotic cells were 
quantitated based on nuclear morphology using fluorescence microscopy and 
the percentage of non-apoptotic cells was calculated. A minimum of 100 
cells was counted for each sample, and each experiment was done at least 
in duplicate. Since a small fraction of cells in any normally growing cell 
culture is undergoing apoptosis, spontaneous apoptosis in untreated or CHX 
alone treated samples was also quantitated. The percentage of 
non-apoptotic cells in the TNF or anti-Fas+CHX treated samples was then 
normalized by correcting for the frequency of spontaneous apoptosis in the 
untreated or CHX alone samples, respectively. 
BJAB cells were grown at 3.times.10.sup.5 cells/ml and treated with 
anti-Fas antibody at a concentration of 250 ng/ml (unless indicated 
otherwise) for 18 hours after which an aliquot was stained with acridine 
orange as described above. Apoptotic cells and non-apoptotic cells were 
quantitated and normalized to untreated samples. Assays were done at least 
in duplicate. 
Secondary assays of cell death used were the MTT conversion assay (as 
described in Opipari, A. W. et al. J. Biol. Chem. (1992) 267:12424-12427) 
and crystal violet staining and were done as described in Tartaglia, L. A. 
et al. (1993) Cell 74:845-853. 
Plasmids, Transfections and Selection of Stably Transfected Lines--The crmA 
gene as shown FIG. 5 was cloned into the pcDNA3 (Invitrogen) mammalian 
expression vector. The crmA gene was obtained from Dr. David Pickup (Duke 
University) and used as a template in a PCR reaction using custom 
oligonucleotide primers with built-in restriction enzyme sites to amplify 
the entire coding sequence. The sequence of the primers are: 
crmA/5'/R1 
5' CAC CGG AAT TCC ACC ATG GAT ATC TTC AGG GAA ATC G 
(Seq. ID. No. 1) 
crmA/3'/XbaI 
5' GCT CTA GAC TCG AGT TAA TTA GTT GTT GGA GAG CAA TAT C 
(Seq. ID. No. 2) 
This PCR fragment was digested with EcoR1 and Xbal restriction enzymes and 
subcloned into the pcDNA3 vector which had been similarly digested. 
Following transformation into competent XL-1Blue host E. coli cells 
(Stratagene), individual colonies were grown up, plasmid extracted and the 
presence of the crmA gene confirmed by both restriction mapping and DNA 
sequence analysis. 
The resulting expression construct or pcDNA3 itself (as the control) was 
introduced into both MCF7 and BJAB cells by electroporation. MCF7 cells 
were electroporated at 330 V, 960 .mu.F in 0.4 cm cuvettes (BioRad), 
plated onto 100 mm dishes at varying dilutions and selected with G418 
sulfate (Gibco-BRL) at a concentration of 500 .mu.g/ml. After selection 
for three weeks, pooled populations from each transfection were prepared 
by trypsinizing dishes containing several hundred colonies. Additionally, 
clonal cells lines were derived by picking individual colonies from 
selected dishes. BJAB cells were electroporated at 220 V, 960 .mu.F in 0.4 
cm cuvettes (Bio-Rad) and selected in 3 mg/ml G418 sulfate. One day 
following transfection, a portion of the cell population was diluted at a 
concentration of 2500 cells/well in 96-well dishes from which clonal lines 
were obtained after G418 selection. The remainder of the cells were 
retained as the pooled population. 
Experiment III 
Cell Lines, TNF and Anti-Fas Antibody--The MCF7 cell line was a 
TNF-sensitive subclone obtained from Dr. David R. Spriggs (University of 
Wisconsin). MCF7 is a breast carcinoma epithelial cell line which 
expresses TNF receptor and is sensitive to TNF killing. The BJAB cell line 
was a gift of Dr. Fred Wang (Harvard). Recombinant TNF (specific activity 
6.27.times.10.sup.7 U/mg) was obtained from Genentech (South San 
Francisco, Calif.). Anti-Fas monoclonal antibody (clone CH-11, IgM) was 
obtained from Pan Vera (Madison, Wis.). 
RNA Isolation and Northern Analysis--RNA isolation and northern analysis 
were carried out as described in Dixit, V. M. et al. (1990). J. Biol Chem. 
265:2973-2978. PCR (Perkin-Elmer) was used to generate a probe spanning 
the coding region of the crmA gene as described above. .beta.-actin cDNA 
probe was purchased from Clontech (Palo Alto, Calif.) and the 
hybridization signal was visualized as a digitized image on a Molecular 
Dynamics Phosphorimager. 
Experiment IV 
Induction of Apoptosis by TNF and anti-Fas--A subclone of the MCF7 breast 
carcinoma epithelial cell line which expressed TNF receptor and was 
sensitive to TNF killing was chosen for these studies. This cell line is 
characterized in Spriggs, D. R. et al. (1988) J. Clinc. Invest., 
81:455-460. Further analysis revealed that Fas was also expressed on these 
cells and that crosslinking with an anti-Fas monoclonal antibody in the 
concomitant presence of the protein synthesis inhibitor cycloheximide 
induced cell death. 
Cycloheximide aline for the duration of the assay did not induce cell death 
beyond the negligible frequency of spontaneous apoptosis which is observed 
in any untreated cell culture. Anti-Fas alone was not cytotoxic, but this 
is not surprising, since induction of cell death in non-lymphoid cells by 
Fas activation has been reported to require the concomitant presence of 
either transcriptional or translational inhibitors. See Itoh, N. et al. 
(1991) Cell 66:233-243. 
A B-cell lymphoma cell line (BJAB) also was examined. It expresses a high 
level of Fas and was killed by the addition of anti-Fas antibody in the 
absence of a protein synthesis inhibitor. 
Cell death can occur by two biochemically and morphologically distinct 
processes: apoptosis and necrosis. In these studies, cell death was first 
confirmed to be the result of TNF or anti-Fas induced apoptosis, not 
necrosis. Although various markers of apoptosis have been reported, the 
phenomenon is preferably defined at the morphological level and is 
characterized by chromatin condensation and margination along the inner 
nuclear membrane, cytoplasmic condensation and membrane blebbing without 
disintegration of the cellular membrane. See Duvall E. et al. (1986) 
Immunol. Today 7:115-119. Necrosis, conversely, is defined by cytoplasmic 
swelling and lysis of the cell membrane and, importantly, does not exhibit 
the chromatin margination characteristic of apoptosis. DNA laddering, 
representative of cleavage at internucleosomal intervals, is seen in some 
but not all forms of apoptosis, further emphasizing the importance of 
morphological criteria in defining apoptosis. See Barres, B. A. et al. 
(1992) Cell 70:31-46. Nuclear morphology of cells dying in response to TNF 
or anti-Fas antibody was examined following staining with the DNA-binding 
dyes propidian iodine (MCF7 cells) and acridine orange (BJAB cells). 
Fluorescence microscopy laser scanning confocal microscopy demonstrated 
marked changes in nuclear morphology in the MCF7 cells in response to 
either TNF or anti-Fas-CHX and in the BJAB cells in response to anti-Fas. 
Chromatin condensation was clearly visible by immunofluorescence 
microscopy in both cell lines and formed the basis for the later assays of 
apoptosis in transfected cell lines (FIG. 1B for BJAB). Confocal 
microscopy confirmed margination along the inner nuclear membrane (FIGS. 
1A and 1B inset). These morphological criteria of apoptotic cell death 
were further confirmed by transmission electron microscopy. The MCF7 cells 
clearly demonstrated chromatin condensation and margination along the 
inner nuclear membrane, cytoplasmic condensation and increased membrane 
blebbing in response to either TNF or anti-Fas+CHX (FIG. 1A). BJAB cells 
treated with anti-Fas antibody demonstrated chromatin margination and 
cellular shrinkage typical of apoptosis in lymphoid cells. Thus, both TNF 
and Fas induced genuine apoptotic cell death in these cell lines. 
Experiment V 
crmA Blocks TNF- and anti-Fas-Induced Apoptosis--To determine whether crmA 
can function to inhibit cytokine-induced apoptosis, MCF7 and EJAB cell 
lines were transfected with either the expression vector pcDNA3 by itself 
or as a crmA expression construct. Expression of the crmA gene was 
confirmed by northern analysis. Stable transfectants were generated by 
neomycin selection, and pooled populations of neomycin-resistant cells 
were assayed for crmA expression. These pooled populations were analyzed 
for their sensitivity to TNF- and anti-Fas-induced apoptosis by direct 
quantitation of apoptotic cells based on nuclear morphology following 
staining with DNA-binding dyes and visualization by fluorescence 
microscopy. Dramatic resistance was seen with either TNF or Fas in both 
cell lines. This was remarkable, given that in the pooled population of 
neomycin-resistant cells transfected with crmA, a significant fraction of 
cells were likely not expressing crmA due to, among other reasons, 
nonproductive integration of the expression construct into genomic DNA. 
In addition to the pools, clonal lines were derived from both MCF7 and BJAB 
transfectants and challenged by activation of the TNF and Fas death 
pathways. In the MCF-7 cell line, vector clones were uniformly sensitive 
to apoptosis induced by either TNF or anti-Fas+CHX, whereas among the 
transfected clones, those which expressed detectable crmA were totally 
resistant to apoptosis (FIG. 3A). Indeed, lines expressing the highest 
levels of crmA were totally resistant to apoptosis (FIG. 3A) and showed no 
morphologic cytopathic effects (FIG. 3B), demonstrating the crmA can 
completely block the TNF- and Fas-mediated death pathways. Similarly, 
among BJAB transfected clones, the crmA expressing the lines were markedly 
resistant to anti-Fas induced apoptosis whereas the vector clones were 
universally sensitive (FIG. 4A). Importantly, in both MCF7 and BJAB 
transfectants, those clones expressing the highest levels of crmA were the 
most resistant while those clones expressing little or undetectable levels 
were the most sensitive (FIG. 3A and FIG. 4A). Although direct visual 
quantitation of apoptotic nuclei is the preferable measure of apoptosis, 
comparable results were obtained when either an MTT-conversion based death 
assay (FIG. 3A insert and FIG. 4A inset) or crystal violet staining (FIG. 
3C) was employed to assess cell survival. 
The dose of death stimulus was increased to determine if protection 
conferred by crmA from cytokine-induced apoptosis could be attenuated. 
Remarkably, crmA afforded comparably high levels of protection from 
anti-Fas-induced apoptosis in response to doses of antibody 250 times 
greater than those needed to kill greater than 95% of the vector 
transfected cells (FIG. 4B). Similar results were obtained when the dose 
of TNF was similarly varied for the MCF7 transfectants, implying that crmA 
is functioning as an exceptionally potent inhibitor of cell death at a 
presumably critical step in the death pathway. 
These results describe an important new function for crmA--the blockade of 
TNF- and Fas-mediated apoptosis. Given the importance of both TNF and Fas 
in the host anti-viral response, it is likely that this function of crmA 
is important for productive viral infection in vivo. crmA represents yet 
another example of viral economy in which two important functions, namely 
the inhibition of IL-1.beta. production and the prevention of apoptosis, 
are embedded in one protein. 
Additionally, this data have implications for the unification of death 
pathways in general. First, the fact that crmA blocks both TNF- and 
Fas-mediated apoptosis, especially in the MCF7 cells that possess both 
receptors, suggests that they signal death through a biochemically common 
pathway. This hypothesis is supported by the finding that the cytoplasmic 
regions of both these receptors encompass a region of homology which has 
been defined by mutational analysis as a "death domain" and which 
presumably interacts with a common set of signal transduction molecules. 
Further, it is not apparent that crmA blocks cell death triggered by two 
very different stimuli: growth factor withdrawal in neuronal cultures and, 
activation of cytokine receptors. It is of note that apoptosis in these 
two systems has been suggested to occur through biochemically distinct 
pathways, in that apoptosis in the former system is dependent on new 
protein synthesis and death is blocked by cycloheximide, whereas in 
contrast TNF- and Fas-mediated cytotoxicity is independent of new protein 
synthesis and is, in fact, enhanced by cycloheximide. Thus, at some point, 
the death pathway in both systems converges upon a crmA-inhibitable step, 
likely the activation of a protease. 
Throughout this application, various publications are referred to by their 
bibliographic citation. The disclosures of these references are hereby 
incorporated by reference into this application to more fully describe the 
state of the art to which this invention pertains. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 4 
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- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 37 base - #pairs 
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(D) TOPOLOGY: linear 
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- # - # - # 
Met 
- # - # - # 
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Asp Ile Phe Arg Glu Ile Ala Ser Ser Met Ly - #s Gly Glu Asn Val Phe 
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393 
Ile Ser Pro Pro Ser Ile Ser Ser Val Leu Th - #r Ile Leu Tyr Tyr Gly 
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441 
Ala Asn Gly Ser Thr Ala Glu Gln Leu Ser Ly - #s Tyr Val Glu Lys Glu 
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489 
Ala Asp Lys Asn Lys Asp Asp Ile Ser Phe Ly - #s Ser Met Asn Lys Val 
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537 
Tyr Gly Arg Tyr Ser Ala Val Phe Lys Asp Se - #r Phe Leu Arg Lys Ile 
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- - GGA GAT AAT TTC CAA ACT GTT GAC TTC ACT GA - #T TGT CGC ACT GTA GAT 
585 
Gly Asp Asn Phe Gln Thr Val Asp Phe Thr As - #p Cys Arg Thr Val Asp 
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633 
Ala Ile Asn Lys Cys Val Asp Ile Phe Thr Gl - #u Gly Lys Ile Asn Pro 
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- - CTA TTG GAT GAA CCA TTG TCT CCA GAT ACC TG - #T CTC CTA GCA ATT AGT 
681 
Leu Leu Asp Glu Pro Leu Ser Pro Asp Thr Cy - #s Leu Leu Ala Ile Ser 
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- - GCC GTA TAC TTT AAA GCA AAA TGG TTG ATG CC - #A TTT GAA AAG GAA TTT 
729 
Ala Val Tyr Phe Lys Ala Lys Trp Leu Met Pr - #o Phe Glu Lys Glu Phe 
130 1 - #35 1 - #40 1 - 
#45 
- - ACC AGT GAT TAT CCC TTT TAC GTA TCT CCA AC - #G GAA ATG GTA GAT 
GTA 777 
Thr Ser Asp Tyr Pro Phe Tyr Val Ser Pro Th - #r Glu Met Val Asp Val 
150 - # 155 - # 160 
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825 
Ser Met Met Ser Met Tyr Gly Glu Ala Phe As - #n His Ala Ser Val Lys 
165 - # 170 - # 175 
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873 
Glu Ser Phe Gly Asn Phe Ser Ile Ile Glu Le - #u Pro Tyr Val Gly Asp 
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921 
Thr Ser Met Val Val Ile Leu Pro Asp Asn Il - #e Asp Gly Leu Glu Ser 
195 - # 200 - # 205 
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969 
Ile Glu Gln Asn Leu Thr Asp Thr Asn Phe Ly - #s Lys Trp Cys Asp Ser 
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#25 
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ACA 1017 
Met Asp Ala Met Phe Ile Asp Val His Ile Pr - #o Lys Phe Lys Val Thr 
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Gly Ser Tyr Asn Leu Val Asp Ala Leu Val Ly - #s Leu Gly Leu Thr Glu 
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1113 
Val Phe Gly Ser Thr Gly Asp Tyr Ser Asn Me - #t Cys Asn Ser Asp Val 
260 - # 265 - # 270 
- - AGT GTC GAC GCT ATG ATC CAC AAA ACG TAT AT - #A GAT GTC AAT GAA GAG 
1161 
Ser Val Asp Ala Met Ile His Lys Thr Tyr Il - #e Asp Val Asn Glu Glu 
275 - # 280 - # 285 
- - TAT ACA GAA GCA GCT GCA GCA ACT TGT GCG CT - #G GTG GCA GAC TGT GCA 
1209 
Tyr Thr Glu Ala Ala Ala Ala Thr Cys Ala Le - #u Val Ala Asp Cys Ala 
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#05 
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GTG 1257 
Ser Thr Val Thr Asn Glu Phe Cys Ala Asp Hi - #s Pro Phe Ile Tyr Val 
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- - ATT AGG CAT GTC GAT GGC AAA ATT CTT TTC GT - #T GGT AGA TAT TGC TCT 
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Ile Arg His Val Asp Gly Lys Ile Leu Phe Va - #l Gly Arg Tyr Cys Ser 
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1357 
Pro Thr Thr Asn 
340 
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1 5 - # 10 - # 15 
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65 - # 70 - # 75 - # 80 
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85 - # 90 - # 95 
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100 - # 105 - # 110 
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115 - # 120 - # 125 
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130 - # 135 - # 140 
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145 1 - #50 1 - #55 1 - 
#60 
- - Val Ser Met Met Ser Met Tyr Gly Glu Ala Ph - #e Asn His Ala Ser 
Val 
165 - # 170 - # 175 
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225 2 - #30 2 - #35 2 - 
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Thr 
245 - # 250 - # 255 
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260 - # 265 - # 270 
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290 - # 295 - # 300 
- - Ala Ser Thr Val Thr Asn Glu Phe Cys Ala As - #p His Pro Phe Ile Tyr 
305 3 - #10 3 - #15 3 - 
#20 
- - Val Ile Arg His Val Asp Gly Lys Ile Leu Ph - #e Val Gly Arg Tyr 
Cys 
325 - # 330 - # 335 
- - Ser Pro Thr Thr Asn 
340 
__________________________________________________________________________