Note: Descriptions are shown in the official language in which they were submitted.
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DIAGNOSIS AND TR~AT~ENT OF
DOUBLE-STRANDED RNA DEFICIENCY STATES
This invention relates to diagnosing a dsRNA deficiency
state and procedures to supply exogenous dsRNA of appropriate
molecular configuration to restore it to a normal level.
New methods in diagnosis and therapy of viral diseases,
chronic pathogen infections generally and cancer emerge when
the diverse roles in host defense played by natural dsRNA are
uncovered. For example, development of AIDS (a retroviral
infection/cancer) is associated with progressively severe
dysfunction in biological processes which I have found to be
catalyzed by dsRNA including not only interferon biosynthesis,
but also bioactive 2-5A production, RNase L activity and various
cell-mediated immune functions. I show that specific reduction
in bioactive dsRNA, or enzymes which depend on dsRNA, within
specific cells contributes to disease progression.
A deficiency state in pathways involving bioactive dsRNA
also lies at the core of various cellular lesions in host
defense systems leading to death. Earlier, I detected an
inhibitor of RNase L in lymphocytes of HIV-infected ARC and AIDS
patients which is just one result (from many) of inadequate
intracellular dsRNA levels. Therapy with appropriately
configured synthetic d~RNA eliminates this inhibitor(s), which
can explain part of the clinical responses I observed with
respect to reduction in virus concentration and re~toration of
immune function in patients with a variety of chronic viremias
which at present continue to largely defy definitive medical
intervention. Accordingly, I have now developed an integrative
invention which can explain, quantitate, and correct a variety
of dsRNA deficiency states in varied disease settings. I have
also discovered that certain dsRNAs, e.g., mismatched dsRNA, can
1 336683
actually substitute for a naturally occuring dsRNA which help
maintain functions essential for normal cell processes. The
absence of such dsRNA regulation, if not corrected with a
suitable exogenous dsRNA, can cause various abnormal cellular
processes to develop. Abnormal cellular processes then lead to
chronic viral or other intracellular pathogen infections,
reduced immune cell functions and, ultimately, chronic morbidity
and possibly death itself.
Clinical Strategies. Viral infections can often follow
partial disturbance of the immune system through agents as
diverse as stress and viruses which directly attack immune
function. For example, AIDS (Acquired Immune Deficiency
Syndrome) follows the progressive deterioration of the immune
system caused by infection of T lymphocytes by human
immunodeficiency virus, HIV (Coffin et al., Science vol. 232,
p. 697, 1986).
For completeness, it should be noted that different designators
for the HIV virus exist; LAV is the designator for the AIDS
virus isolated at the Pasteur Institute, Paris, France and
HTLV-III is the designator for the AIDS virus isolated at the
National Institute of Health, Bethesda, Maryland, U.S.A.
Currently, HIV is used as a generic term for the virus.
Frequently in this text, the HIV virus will be referred to
generically or designated HIV or HTLV-III or LAV without
intending to differentiate between them. Furthermore, the term
HIV in the specification and claims includes all other viruses
which may be associated with producing AIDS, whether yet
isolated or not.
HIV infection is a progressive disease although the rate of
transition from one phase to another is variable. Asymptomatic
individuals infected by HIV can develop a condition (LAS or
pre-ARC) characterized by lymphadenopathy. Patients progress to
AIDS-related complex or ARC by exhibiting the T4 cell deficit
and later show reduced ability of lymphocytes to undergo
antigen-stimulated proliferation and IFN-gamma production.
These patients lose various cell mediated immune functions such
1 336683
as delayed cutaneous hypersensitivity, eventually becoming
complete ly anergic. They exhibit evidence of breakdown in host
defense mechanisms including Herpes Zoster infections, oral
candidiasis (thrush), and symptoms such as prolonged fevers,
night sweats diarrhea and weight loss. Finally, these patients
experience certain severe opportunistic infections (e.g.,
Pneumocystis carinii pneumonia) and are then defined as having
full-blown AIDS, with an approximately 50% mortality within 12
months. The HIV virus infection is chosen as an illustrative
case simply because the level of dsRNA deficiency is so
pronounced. Many other diseases, including indolent viral
infections, will be associated with similar deficiencies; thus,
my invention has unexpected wide applicability.
Importantly, the typical progression outlined above does
not describe the remarkable disparity in clinical symptomatology
among HIV-infected patients or many other patients with -chronic
disease such as Epstein Barr or EB infection, hepatitis virus
infection, cytomegalovirus, herpes infections, etc. Components
of the immune system decay with vastly different rates in
various patients and the lack of bioactive dsRNA is a cause or
contributing factor in this process. HIV infection may develop
in a variety of different types of cells (e.g., blood cells,
glial cells). Some patients develop neurological dysfunction as
their first symptom. Thus, various individuals may have vastly
different therapeutic requirements even though all will probably
require HIV eradication from T4 cells.
Two general pathological features need to be addressed in
many different viral infections including those of ARC pa-
tients: a collection of immune deficits and ongoing viral infec-
tion (reviewed in Fauci, Proc. Nat'l. Acad. Sci. USA, vol. 83,
p. 9278, 1986). Therapeutic intervention which attacks only the
immune deficits but fails to control virus replication appears
to be counterproductive especially when such treatment activates
T4 cells. For example, T-helper cells with Tac receptors are
responsive to the lymphokine IL-2. In AIDS victims, IL-2 causes
expansion of HIV-sensitive cells and apparent worsening of the
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di6ease.
Attacking viral infections directly, e.g., the HIV infec-
tion, has met with some success especially evidenced by the
viral reverse transriptase inhibitor designated azidothymidine
(AZT) which can reduce HIV "load" in some patients and prolong
life. Such therapy i8 not without undesirable results. In a
minority of patients, AZT al~o causes temporary recovery of
T-cell meditated i~mlln;ty as measured by a transient rise in T4
cells and by appearance of delayed type hypersen~itivity (Fischl
et al. New Eng. J. Med., July, 1987) However, reverse
transcriptase inhibitors are toxic at doses needed to produce
significant antiviral effects and are thus poorly tolerated over
time. Antiviral agents may need to be administered for the life
of the patient.
IFN is both ineffective as a general antiviral compound and
as an anti-AIDS drug, probably because it is not an antidote for
the dsRNA-relted defects which I have now uncovered. Certainly,
ARC/AIDS victims have various "defects" in IFN pathways, such as
the production of a defective, acid-labile IFN and inability of
T4 cells to produce appropriate amounts of IFN gamma which are
well described in the medical literature. Earlier, I concluded
that these various IFN-related defects in ARC and AIDS patients
may prove to be relatéd in whole or part to a lack of normal
RNase L (a terminal pathway mediator) activity in lymphocytes.
Although dsRNA-dependent 2-SA syntheta~e i~ elevated in blood
lymphocytes of individuals with ARC/AIDS as well as other
chronic viremias, I have found that the same blood cells show
diminished levels of authentic 2-SA and low RNase L ~see
below). I correlated the low RNase L activity with low guanti-
ties of Mr80,000 protein capable of binding a photoaffinity
labeled analogue of 2-SA. My f indings are consistent with a
dsRNA pathway(s) which has ~have) been shut down and the
relative absence and/or inhibitor of RNase L.
Vaccination against HIV and other viruses, and the use of
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passive anti-HIV antibodies, are also under consideration for
viral prevention and treatment, respectively, but may have sig-
nificant limitations: for example, HIV (as well as influenza
virus in particular) readily can infect cells covertly. I also
described a role for dsRNA to enhance antibody formation against
viruses in general and retroviruses in particular in the
above-mentioned patent application.
I describe herein a new phenomenon that argues for
double-stranded RNAs (dsRNA) to constitute the first group of
molecules which are effective against both the viral and
immune lesions of various subacute/chronic viral infections
such as HIV and others. These defects can be corrected with
laser-like precision without adversely affecting other bodily
functions which is a common limitation of presently available
therapies for chronic viral infections. I conclude that
patients with unresolved and indolent viral infections often
have specific defects in vital intracellular dsRNA-dependent
pathway~ which can be reversed or ameliorated at least in part
by an exogenously supplied dsRNA (this is explained
graphically in the flow chart of Figure 1), and that monitoring
these pathways, before and during treatment, provides a novel
efficacious manner to gauge the degree of dsRNA replacement
therapy required an individual patient or specific disease
basis.
Critical enzymes associated with host defense require
dsRNAs for expressing bioactivity. These enzymes can be activat-
ed by exogenous dsRNA if the level of natural intracellular
dsRNA is too low. Part of the pleiotropic action of dsRNA de-
rives from its ability to induce the synthesis of the entire
range of IFNs - alpha, beta and gamma - which operate through a
group of intracellular mediators including a dsRNA-dependent
protein kinase, dsRNA-dependent 2-5A synthetase and 2-5A-depen-
dent RNase L. These dsRNA-dependent enzymes may carry out many
biological activities attributable to IFN as reviewed by Lengyel
(Ann. Rev. Biochem., vol. 51, p. 251, 1982). For example,
production of an antisense RNA which blocks expression of
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2-5A-synthetase causes profound sensitivity of animal cells to
infection by different viruses (Bendetti et al. Prac. Natl.
Acad. Sci USA, vol. 84, p. 658, 1987). The dsRNA-activated
protein kinase phosphorylates elF2 which inhibits protein
syntheses; it also has proteolytic activity to degrade viral
proteins. DsRNA-activated 2-5A synthetase synthesizes 2-5A
which activate RNase L, a pathway which I suggest plays a
crucial role both in the inhibition of various animal viruses
and in cell growth control (escape from which may lead to tumor
formation). 2'-5' oligoadenylic acids are unusual in that the
normal 3',5' phosphodiester linkage characteristics of most
dsRNA has been replaced by a novel phosphodiester bond with
acquisition of new biological properties.
Antiviral Activity of dsRNA. dsRNA is a well-known
inducer of an antiviral state (Marcus et al, Nature, vol. 66,
p. 815, 1977). Similarly, poly(A):poly(U) generated an
antiviral state without inducing IFN. But, prior to my current
invention, it was not known that natural dsRNA regulators exist
and, therefore, that actual dsRNA deficiency states are common
in humans, particularly associated with pathogenic episodes
(viral, fungal, protozoan, bacterial invasions, etc., especially
those which have intracellular (human) presence as a valuable or
necessary components of their life cycle).
Antiproliferative Activity of dsRNA. Earlier, I found
(J. IFN. Res., vol. 6, p. 373, 1986) that 42% of more than 100
fresh human tumor specimens, when studied in soft agar, showed a
50% or greater reduction in tumor cell colony formation after
only one exposure to dsRNA. These cells are dsRNA deficient. I
also described independent IFN and dsRNA sensitivity in various
human tumor cell lines and found that human tumors propagated in
nude mice were sensitive to dsRNA; dsRNA therapy was curative of
some tumors (e.g., renal) and the animals lived to a normal life
expectancy (see published European patent application
0,113,162). Herein, I report a strong correlation between
clinical responses and dsRNA-dependent enzyme activation as
opposed to detectable IFN or IL-2 induction secondary to dsRNA
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administration.
Immune ~nh-ncement Activity of dsRNA. Earlier I reported
(J. Immunol., vol. 124, p. 1852, 1980) that dsRNA increased
human natural killer NK cytlytic activity against human leukemia
cells. The dsRNA structural requirements for NK augmentation
paralleled those needed for 2-5A synthetase activation and for
IFN induction.
Gene Control Mediated by d~RNA. Sullo et al (Cell,
vol. 43, p. 793, 1985) demonstrated that dsRNA induces compe-
tence genes in resting fibroblasts including the oncogenes desig-
nated fos and mYc. As used herein, "competence genes" are
those genes in a cell whose actions are required in order to
modulate vital actions such aq multiplication, growth, etc. The
IFN-beta gene induction by dsRNA may utilize a regulatory pro-
tein which is removed upon exposure of cells to dsRNA (Zinn et
al. Cell, vol. 45, p. 611, 1986). I have determined that, to
biological activity, exogenously supplied dsRNAs mimic a natu-
ral, nonviral, intracellular dsRNA such as heterogeneous nuclear
RNA or mRNA complexed with poly(U). For example, I recently
discovered an IFN-inducible dsRNA in Hela cell nuclei capable of
activating 2-5A synthetase i vitro. The advent of cellular
immunity far back in the evolutionary cycle in animals apparent-
ly set the stage for the evolution of IFN/dsRNA molecules toward
implementing cellular differentiation, particularly differentia-
tion promoted by IFN, I was able to achieve the more general
insight that dsRNA can implement differentiation promoted by
other regulatory proteins which set the stage for my definition
of dsRNA deficiency state~.
In the ~c~c~."~,,ying drawings:
Figure 1 is a flow chart illustrating the dual pathways to
host morbidity and the pathway to host recovery;
Figure 2A i9 a graph plotting the number of days prior to
and after initiation of IFN therapy in the amount indicated
against syntheta~e activity;
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Figure 2B is a graph compàring white blood cell count and
quantity of mismatched dsRNA after 120 days of treatment;
Figure 3 is a photograph of polyacrylamide gel
electrophoresis plates showing various zones and bands along
each track; and
Figure 4 is a graph comparing absolute number of T4
lymphocyte levels per cubic millimeter of blood, expressed as %
change from baseline, to months of therapy.
I describe herein a new phenomenon that argues for
double-stranded RNAs (dsRNA) to constitute the first group of
molecules which are effective against both the viral and
immune lesions of various subacute/chronic viral infections
such as HIV and others. These defects can be corrected with
laser-like precision without adversely affecting other bodily
functions which is a common limitation of presently available
therapies for chronic viral infections. I conclude that
patients with unresolved and indolent viral infections often
have specific defects in vital intracellular dsRNA-dependent
pathways which can be reversed or ameliorated at least in part
by an exogenously supplied dsRNA (this is explained graphically
in the flow chart of Figure 1), and that monitoring these
pathways, before and during treatment, provides a novel
efficacious manner to gauge the degree of dsRNA replacement
therapy required an individual patient or specific disease
basis.
This invention provides a diagnostic procedure for
determining a dsRNA deficiency state in a patient's sample,
quantifying this deficiency (if any), and providing a
therapeutic procedure for restoring any dsRNA deficiency
identified by the administration of exogenous dsRNA optionally
in combination with a lymphokine. Typically, a dsRNA deficiency
state is evidenced within a component cell of the immune system
whether or not the component cell i8 in the process of
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multiplication or differentiation. DsRNA deficiency is
evidenced, for example, by the inability of a component cell of
the immune system to maintain a sufficient level of bioactive
RNase L. Monitoring of the intracellular dsRNA-dependent
pathways before, during and after therapy enables the clinician
to guage the degree of dsRNA replacement therapy required on an
individual basis. Other means to assess dsRNA deficiencies are
given in the detailed description that follows. "Mediators"
are herein operationally defined as any intracellular moieties
(e.g., specific oligonucleotides, proteins, dsRNAs - alone or in
combination) which effect specific biochemical functions
initiated by presence of dsRNA. Such biochemical functions will
contribute to a strengthening of host defense mechanism(s) at
either the single cell or whole (organism) level.
Conditions susceptible to the therapeutic procedures of
this invention are generally those in which a deficiency of the
intracellular dsRNA level is below normal limits, as compared
with healthy individuals, dsRNA deficiency causing tissue pathol-
ogy and/or in the presence of an abnormally low dsRNA level
coupled with or coexisting with the presence of an intracellular
pathogen. More specific conditions or tissue pathologies and
constitutional symptoms include viral infections such as
retrovirus infections including HIV, herpes family virus,
paranyxovirus, rhinovirus, hepatitis and chronic fatigue
syndrome. Others include uncontrolled proliferation of cancer
cells and pathologies of the immune system whether or not the
component cell is in the process of multiplication or
differentiation.
By "mismatched dsRNAs" are meant those in which hydrogen
bonding (base stacking) between the counterpart strand is rela-
tively intact, i.e., is interrupted on average less than one
base pair in every 29 consecutive base residues. Mismatching is
an interruption of the normal geometry of the RNA double helix
by in-pouching (or out-pouching) of the strands which represent
points of vulnerability of the dsRNA to digestion by
ribonucleases. The term "mismatched dsRNA" should be understood
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accordingly.
The dsRNA may be a complex of polyinosinate and a
polycytidylate containing a proportion of uracil bases or
guanidine bases, e.g., from 1 in 5 to 1 in 30 such bases
(poly I. poly (C4 29 x > U or G).
The dsRNA may be of the general formula
rIn.r(C12U)n. Other suitable examples of dsRNA are
discussed below.
In the preferred mismatched dsRNA, rIn-r(C12,U)n, a
region consisting of an uninterrupted stretch of 6 to 12 base
pairs, i.e., one-half to one full turn of an RNA helix, serves
both as biotrigger causing release of lymphokines and as an
obligate intracellular co-factor for enzymes comprising the
natural antiviral pathways. The mismatched regions consisting
of uracil residues is periodically inserted in the
polypyrimidine strand to accelerate dsRNA hydrolysis and thus
prevent toxicity.
The mismatched dsRNAs preferred for use in the present
invention are based on copolynucleotides selected from poly
(Cn,G) in which n is an integer having a value of from 4 to
29, and are mismatched analogs of complexes of polyriboinosinic
and polyribocytydilic acids, formed by modifying rIn.rCn to
incorporate unpaired ba~es (uracil or guanidine) along the
polyribocytidylate (rCn) strand. Alternatively, the dsRNA may
be derived from poly (I). poly (C) dsRNA by modifying the
ribosyl backbone of polyriboinosinic acid (rIn) e.g., by in-
cluding 2'-0-methyl ribosyl residues. These mismatched analogs
of rIn.rCn, preferred ones of which are of the general formu-
la rIn.r(C11_14,U)n and rIn.r(C29,G)n, are described
by Carter and Ts'o in U.S. Patents 4,130,641 and 4,024,222.
The
dsRNAs described therin generally are suitable for u~e according
to the present invention. In certain instance~, complementary
oligonucleotide duplexes (helices) will also suffice as
replacement therapy.
Other examples of mismatched ds~NA for use in the invention
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include: -
poly (I) poly (C4,U)
poly (I)-poly (C7,U)
poly (I) poly (C13,U)
poly (I)-poly (C22,U)
poly (I) poly (C20,G)
poly (I)-poly (C29,G) and
poly (I)-poly (Cp) 23 G~p
dsRNA-Lymphokine ~ynergy. Since dsRNA exerts a signifi-
cant component of its biological action through a specific
lymphonine system mediated in part by IFN, I reasoned that it
must (a) induce IFN in IFN-deficient cells as well as (b) acti-
vate relevant dsRNA-dependent enzyme mediators, the cellular
dsRNA being non-inducible in cells failing to respond to
exogenously applied IFN.
If dsRNA operates solely thought the IFN sy~tem and if
dsRNA is in excess in cells, then an excess of IFN alone would
exert as much bioactivity as the combinations of IFN and dsRNA.
However, this must be a relatively rare condition since I have
now found many examples of dsRNA-IFN synergy and a paucity of
examples of no synergy. For example, I found that dsRNA was
synergistic with human IFN alpha, beta or gamma in inhibiting
the growth of cells form human bladder carcinoma, lung
carcinoma, and fibro ~ ~l~. I extended these results to 15
other human tumor cell lines with only one line failing to show
antiproliferative synergy. I observed similar antitumor syner-
gism was seen in some human tumor engrafted in nude mice and,
clinically, I consistently observed that the combination of IFN
and dsRNA was superior to either agent alone as an anticancer
regimen in both kidney tumors and CML.
dsRNA Deficiency States. As a prototype pathway, the
2-5A synthetase/RNase L pathway includes one or more dsRNAs,
2-SA, linked enzymes and inhibitors. There exists a direct
biochemical connection between dsRNA and RNase L in that 2-5A,
the product of dsRNA-dependent 2-5A synthetase, is a necessary
1 336683
~
cofactor for RNase L activity. The examples below are intended
to exemplify a few of many possible kinds of defects in
dsRNA-mediated pathways which can be reversed by exogenously
supplied dsRNA.
The flow chart of Figure 1 further illustrates the relation-
ship between dsRNA sufficiency, leading to host recovery, and
dsRNA deficiency, leading to host morbidity. Bioactive dsRNA is
produced intracellularly or is introduced by certain events
which lead to an antiviral state as well as immune cell differ-
entiation and host recovery.
The in vitro measurement of 2-5A synthetase is of
limited value, since this assay does not reflect the in vivo
activity of the 2-5A synthetase. Therefore, I have developed a
method to extract and ~uantitate the 2-5A from the peripheral
blood mononuclear cells (PBMC) of healthy individuals and from
pathogen-infected patients before and after mismatched dsRNA
therapy. The concentration of 2-5A can be determined by the
ability of the 2-5A to activity affinity-purified RNase L to
degrade poly(U)[32P]pCp.
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TABLE 1
I~t 11~1 2-5A r, I.~lion and In ~itro ~ of 2-5A
1 1 and eNh ~ L in PB~C Eal L. oF ~iral-T F_ I - T '',' ' 1.
PBMC Week~ on 2-5A Ifil~ lar RNa~e L
~our~e ~ ' ,y.. t' t Z-5A
d~RNA activity latent activated
colu~n. 1 2 3 4 5
~1 A m S 0 49 <0.2 260
~ 4 105 N
KS 9 4 <0.2
~5 10 1.8
19 21 0.8
24 26 >10.0
28 20 >10.0
~2 A m S -1 205 <0.2 240
~ O ~80 ND
KS 4 117 1.4
~ 8 19 2.4
PCP 21 ND 6.8
26 9 5.4
~3 ARC -6 11 <0.2 270
2 13 N
4 19 <0.2
17 3 1.8
K ARC 0 130 <0.2 270
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2 185 ND
4 18 0.4
8 ND <0.2
22 ND >10.0
~5 LAS O 30 0 . 6 280 - -
4 36 c0.2
9 12 1.0
17 21 3.0
Healthy 5 0.3-1.1 1350
Vsl l~r ~
a nmole ATP incorporated into 2-5A/mg protein as determined
in poly(I):poly(C)-agarose assays; standard deviation 9%; ND,
not determined.
nmole/g protein as determined in core-cellulose assays (in
duplicate); standard deviation 20%.
c dpm/50 ~g protein as determined in radiobinding assays (in
duplicate) (column 3); standard deviation 20%.
d determined in rRNA cleavage assays (columns 4 and 5) in two
independent assays. Specific cleavage product (SCP) formation
was quantitated by densitometric tracings of photographs of gels
and is expressed as a ratio of SCP formation divided by 18S and
28S rRNA x 100; -, 0; +, 10-30; ++, 31-63; 64-85; ++++, 86-100.
Methods. Heparinized peripheral blood was obtained from
ten healthy individuals and from five homosexual male patients
with LAS, ARC and AIDS (as defined by the Centers for Disease
Control). Clinical, virological and immunological features of
patients involved in this study (patients #1-5) were
14
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characterized previously as patients 10, 8, 1, 7 and 2,
respectively in my Lancet June 6, 1987 article. PBMC were
isolated on Ficoll-Hypaque. L929 and HL929 (an RNase
L-deficient subclone of L929) cells were maintained in monolayer
culture. Cytoplasmic extracts from L929 and HL929 cells were
prepared in glycerol buffer and PBMC extracts were prepared in
NP40 lysis buffer. Protein concentration of the extracts ranged
from 10 - 15 ~g/~l. KS, Kaposi's sarcoma; PCP, Pneumocystis
. . .
carnll pneumonla.
Activity of 2-5A synthetase (column 1) was determined in
PBMC extracts (50 ~g protein/assay) using
poly(I):poly(C)-agarose. 2-5A was isolated from the
ethanol-soluble fraction of PBMC extracts (100 ~g protein) (70%
ethanol, v/v). The ethanol-extracted 2-5A was then lyophilized
and redissolved in water (20 ~1). Concentrations of
intracellular 2-5A present in PBMC extracts (column 2) were
determined in core-cellulose assays with L929 cell extracts as
the source of RNase L from calibration curves obtained with
authentic 2-5A. Activation of RNase L by 2-5A in this assay is
based on the conversion of poly(U)[32P]pCp to acid-soluble
fragments. In control experiments (in the absence of 2-5A),
6500 dpm was retained on glass fiber filters from a total of
17700 dpm of poly(U)132P]pCp added (1.3 ~Ci/nmole). In the
presence of <1 x 10 10 M authentic 2-5A, poly(U)l32P]pCp
was degraded 0%; in the presence of > 1 x 10 7 authentic 2-5A,
poly(U)[32P]pCp was degraded 100%. Latent RNase L levels were
measured by two methods: (i) in radiobinding assays after the
addition of 20,000 dpm of p3A4[32P]pCp to PBMC extracts
(50 ~g protein/assay) (column 3) and (ii) in ribosomal RNA
cleavage assays in the presence of 2-5A (column 4). Extracts
(150 ~g protein/assay) prepared from L929 cells were incubated
in the presence of 5 ~l of ethanol-soluble fractions of PBMC
extracts and 1 x 10 8 M p3A3 in a final volume of 20 ~l
for 60 minutes at 30C. The total RNA was extracted, denatured
and analyzed by electrophoresis on 1.8% agarose gels. The
ethidium bromide stained RNA bands were visualized under UV
1 336683
light. Activated RNase L levels (column 5) were determined by
ribosomal cleavage assays in the absence of added 2-5A.
The concentration of functional 2-5A in PBMC extracts
varied from 0.3 to 1.1 nmole/g protein for healthy humans and
from below detectable levels to 0.6 nmole/g protein in PBMC
extracts from virus-infected patients before mismatched dsRNA
treatment (Table 1, column 2). However, 2-5A accumulated to
greater than 10.0 nmole/g protein as mismatched dsRNA therapy
progressed. 2-5A activated, partially-purified RNase L has
been previously reported to preferentially cleave polu(U), but
not poly(C). The isolated human 2-5A, like authentic 2-5A,
could activate affinity-purified RNase L from either L929 cells
or from PBMC of healthy humans to specifically degrade poly(U);
see Table 4. It is important to note that when TCA is used to
extract 2-5A from PBMC, a poly(C) degrading activity was
extracted in addition to the poly(U) degrading (RNase L)
activity. This poly~C) degrading TCA-extractable activity could
be eliminated by protease digestion or further ethanol
extraction. The poly(C) degradative activity was found in
TCA-soluble fractions of PBMC from all hl~m~ns, but in none of
the permanent cell lines (i.e., HeLa L929, HL929) tested thus
far. The poly(C) degrading activity was not detectable in any
16
1 3 3 6 6 8 3
of the ethanol-soluble fractions of PBMC.
TABLE 3
Effect of dsRNA on Infectiou~ Center Formation by
Respiratory Syncitial Virus-Infected CCL 25 Cell3
C~ ... l,dlion of Infectious Centers Percent
mismatched dsRNA Plate Counts Mean Counts Reduction
0 ~9/~1 49, 44, 15 , 33 42.0
0.5 ~g/Dl 38, 36, 26, 16 33.3 21.7
1.0 ~g/~l 54, 44, 42, 53 48.5 15.5
2.5 ~g/~1 37, 32, 24, 30 30.8 26.7
5.0 ~lol 27, 14, 17, 18 19.0 54.8
10.0 ~g/~l 1, 1, 7, 5 3.5 91.7
25.0 ~glml 0, 2, 0, 0 0.5 98.8
Data not used to calculate mean counts.
The highest concentration of mismatched dsRNA [rI-r(C12,
U)n, 25 ~g/ml] was non-toxic to CCL 25 cells.
(a) Quadruplicate wells of confluent CCL 25 cells in 24
well plates were re-fed with 1.5 ml maintenance medium
containing dsRNA at concentrations indicated above 1 hour prior
to plating of RSV-infected cells. Mock-treated cells were
re-fed dsRNA-free maintenance medium.
(b) 4 x 104 CCL 25 cells in suspension were infected in a
total volume of 1 ml containing 5 x 105 PFU of RSV (M~I
= 12). Viral adsorption occurred during a 2 hour incubation at
37C with intermittent resuspension of the cells. At the end
of this period, the suspension was diluted 1/lO0 in maintenance
medium. 0.5 ml aliquots of the infected cell suspension were
added directly to each experimental well, and to each infectious
center-control well. 0.5 ml volumes of maintenance medium were
added to cell control wells. All cultures were incubated at
1 336683
37C in 5% C02-95% air for 72 hours, fixed with methanol and
stained with Giemsa. Plaques (Infectious Centers) were
counted microscopically.
moi refers to multiplicity of infection; that is, the
approximate number of viral particles available to infect each
target cell. PFU refers to plaque forming units; that is, the
number of viral units as determined by counting the cytopathic
(dead cells).
Although there have been reports on the presence of 2-5A in
tissues isolated from untreated animals as well as the
accumulation of 2-5A in tissues from mice treated with dsRNA,
mine is the first report of the accumulation of 2-5A in a human
tissue. It is well established that authentic 2-5A binds to and
activates RNase L to degrade rRNA to highly characteristic
specific cleavage products (SCP). Therefore, I characterized
the activity of the 2-5A isolated from PBMC extracts in rRNA
cleavage assays. The specific cleavage pattern generated by
RNase L with ethanol-extracted 2-5A was the same as that
obtained in the presence of authentic 2-5A. These results show
that i.v. administration of mismatched dsRNA to virus-infected
patients caused dramatic increases of 2-5A in blood cells
beginning from concentrations which were below detection.
RNase L Activity
The absence of RNase L activity in HIV-infected individuals
was confirmed and extended (Table 1, columns 3, 4, 5). A
sensitive technique based on the cleavage specificity of 2-5A
activated RNase L on rRNA (Wreschner et al, Nucleic Acids
Res., vol. 9, pp. 1571-1581, 1981) permitted the measurement of
activated RNase L in extracts of PBMC isolated from as
little as one ml of peripheral blood. Extracts of an RNase
L-deficient subclone of the L929 cell line (HL929) were utilized
as the source of ribosomes. PBMC extracts of HIV-infected
1 336683
patients had 5-7 times lower levels of latent RNase L than those
of healthy individuals as determined in radiobinding assays
(Table 1, column 3; 240-280 dpm vs. 1350 dpm). Similar
observations have been reported by Wu et al (AIDS Research,
vol. 2, pp. 127-131, 1986). The application of the radiobinding
assay is limited with respect to the determination of latent
RNase L level in patients treated with mismatched dsRNA because
accumulated 2-5A present in PBMC samples from these patients
(Table 1, column 2) can compete for binding to RNase L.
Therefore, determination of the level of the 2-5A-activated
RNase L was essential.
First, levels of activated RNase L were determined by
measuring the specific cleavage of rRNA by PBMC extracts in the
absence of exogenously added 2-5A (Table 1, column 5). Using
this assay, I demonstrated that RNase L was not activated in
PBMC extracts of HIV-infected patients before therapy
(polyacrylamide gels shown in Figure 3). Knowing that the
levels of intracellular 2-5A were low in these PBMC samples
(Table 3, column 2), this result was not unexpected. However,
extracts of PBMC from all healthy individuals tested showed the
presence of activated RNase L which produced specific cleavage
products in rRNA cleavage assays, even though 2-5A in some
healthy individuals was as low as 0.3 nmole/g protein (Table 3,
column 5). It is significant that the absence of RNase L
activity in samples from HIV-infected patients before therapy
cannot be due to accumulation of a competitive inhibitor of 2-5A
as reported (Caylet et al European J. Bioch., vol. 143, pp.
165-177, 1984) RNase L activity could not be restored by the
addition of authentic 2-5A (Table 1, column 4).
Next, levels of latent RNase L were determined by two
independent measures. Latent RNase L activity was determined in
the rRNA specific cleavage assay, but in the presence of added
2-5A (Table 3, column 4). In samples taken before mismatched
dsRNA therapy, no latent RNase L activity could be detected in
any of the 5 patients examined.
The technique of photoaffinity labeling with
19
1 336683
p3A4[32PlpCp was used to determine further if RNase L was
missing or altered in PBMC of HIV-infected patients. Previous
photoaffinity labeling studies have identified a protein of
Mr80,000 with binding affinity for p3A4[32P]pCp and
specific poly(U) endoribonucleolytic activity as RNase L.
Affinity labeling of RNase L present in PBMC extracts was
determined using p3A4[32P]pCp. RNase L from PBMC extracts
(50~g protein) from a normal individual was compared with an ARC
patient (patient before mismatched dsRNA therapy) or L929 cell
extracts (50~g protein) was photolabeled after the addition of
p3A4[32]pCp (30,000 dpm, 3000 Ci/mmole) in the absence or
the presence of authentic p3A4 (1 x 10 8 M). RNase L from
PBMC extracts from a normal individual (lOO~g protein) was
purified on 2-5A core-cellulose and photolabeled after the
addition of p3A4[32P]pCp (30,000 dpm; 3000 Ci/mmole).
After incubation at 0C for 90 minutes, the samples were
transferred to ice-cold porcelain spot plates and photolysed for
3 minutes, using 254 nm UVG-11 Mineralight lamp (Ultraviolet
Products) at a distance of 2 cm (1.0 J/m2). The positions of
the protein markers and the Mr80,000 RNase L were determined.
My photolabeling studies revealed that p3A4[32P]pCp
was covalently linked to a protein with an Mr of 80,000 in
extracts of PBMC from healthy humans (polyacrylamide gel data
not shown). However, no protein was labeled in extracts of PBMC
from a chronically virally-infected patient. Under identical
experimental conditions, a protein of Mr 80,000 was
specifically photolabeled in L929 cell extracts. Addition of
p3A4 (1 x 10 8 M) to incubation mixtures containing the
~q
p3A4[~P]pCp prevented photolabeling thus providing me
additional evidence that the photolabeling was highly specific
for RNase L. Photolabeling studies with the protein purified
from PBMC extracts from a healthy human by the core-cellulose
method revealed covalent linkage to a protein of Mr80,000,
which was capable of degrading poly(U), but not poly(A), poly(C)
nor poly(G), in the presence of authentic 2-4A (Table 4). Taken
together, these results confirm that the protein purified from
1 336683
PBMC of healthy individuals and photolabeled by
p A [32P]pCp
After 4 to 17 weeks on mismatched dsRNA therapy, activated
RNase L was first detected in PBMC extracts from all five
chronically infected patients; by 17 to 28 weeks of mismatched
dsRNA therapy, activated RNase L activity for all five patients
was equivalent to RNase L levels observed in healthy individuals
(Table 1, column 5). The level of activated RNase L showed very
close correlation with the concentration of functional 2-5A
isolated from the same samples (Table 1, as compared with
columns 2 and 5). Of utmost interest, patient #l maintained an
elevated intracellular 2-5A level and fully activated RNase L at
28 weeks, 3 weeks after mismatched dsRNA therapy was
discontinued (Table 1, columns 2 and 5).
The experiments described here show that five HIV-infected
patients possess a common molecular phenotype in blood
mononuclear cells: reduced levels of 2-5A and absence of detect-
able RNase L activity. Blood mononuclear levels for healthy
individuals exhibited a different phenotype in that 2-5A
levels were higher, on average, and RNase L activity was readily
detectable. The latter phenotype is more consistent with that
expected of a functional 2-5A synthetase/RNase L pathway. In
normal individuals, the steady state pool of 2-5A is detectable;
an appreciable fraction of this intracellular 2-5A may become
tightly associated with RNase L, thereby activating the RNase
L. In the experimental procedures elucidated herein, it is
clearly established that in blood mononuclear cells of chronic
virally-infected persons, the intracellular levels of 2-5A are
below detection and there is no activated RNase L as determined
by presently available technology.
In summary, an important discovery here was that defects in
the 2-5A synthetase/RNase L pathway were reversed by therapy
with mismatched dsRNA. My results are not explained simply by
normal levels of RNase L protein without 2-5A, since RNase L
activity in vitro was not restored by adding 2-5A to PBMC
extracts. RNase L protein must be absent or inhibited, a
1 336683
conclusion confirmed by lack of binding by a
photoaffinity-labeled analog of 2-5A. In the treated patients
described here, HIV RNA levels in PBMC were reduced within 10-20
days and the number of infections centers (co-cultivation) of
HIV-infected PBMC declined more slowly. The increase in
intracellular 2-5A and RNase L activity determined here more
closely parallel the loss of infectious centers in these
patients. My results clearly show that the 2-5A
synthetase/RNase L pathway became more active in treated
HIV-infected patients than in healthy humans after several weeks
of therapy with mismatched dsRNA. My results are thus fully
consistent with the hypothesis that certain chronic viral
infections represent a dsRNA deficiency state which can be
reversed by supplying exogenous source of bioactive dsRNA.
The invention is further described with reference to the
following illustrative examples in which all parts and
percentages are by weight unless otherwise indicated.
EXAMPLE 1 - Coordinated IFN and dsRNA Treatments
A dsRNA-deficiency state is one in which IFN-treated tumor
cells show little or no IFN-induced growth arrest. This is a
functional or operational test of dsRNA deficiency which can be
confirmed rigorously if need be by various biochemical measure-
ments described below. In many instances, a clinical diagnosis
of a "dsRNA deficiency" will become sufficient as my invention
becomes more widely practiced. IFNr tumor cells are known to
be rarely deficient in IFN receptors and may contain high or low
levels of mediators, but in either case I find that they respond
to exogenously supplied dsRNA. These examples are of clinical
relevance since they indicate that dsRNA will consistently ex-
pand the therapeutic range of IFN in particular and also other
lymphokines as well, e.g., IL-2, various colony stimulating
factors and TNF. Chronic myelogenous leukemia (CML) is a case
in point. About 60% of CML patients respond to IFN alone, while
40% do not. The latter fail to induce 2-5A synthetase in blood
1 336683
mononuclear cells (MNC) as described by Rosenblum et al. in
Cancer Res., vol. 46, p. 4848, (1986). Figure 2-A presents an
IFN-resistant case which I treated with mismatched dsRNA plus
IFN following a short course (7 days) of IFN alone then with
mismatched RNA in combination with IFN. This patient failed to
induce 2-5A synthetase activity while receiving IFN alone but
did show a substantial increase in activity of this mediator
with the combination of IFN and mismatched dsRNA. Induction of
2-5A synthetase was accompanied by complete hematologic remis-
sion which has lasted for several months, after a transient
increase in blood cell number caused by interrupting prior
chemotherapy. The activity of RNase L was also measured and
found to be 10 to 100-fold higher than normal (Figure 3A, lane
1) yielding RNA cleavage products i vitro which represented
further degradation beyond the usual rRNA cleavage products of
normal RNase L. RNase L activity returned to normal levels and
cleavage profiles after 30 days of therapy with the IFN/dsRNA
combination (Figure 3A, lane 2). I note here that this kind of
response was observed regularly.
EXAMPLE 2 - Viral Infection of T4 Cells
Another dsRNA-deficiency state is one where the animal
virus itself (or its replicative intermediates) supply an
inhibiting dsRNA or a partly bioactive dsRNA for mediator
activations, even though IFN and its mediators are induced by
virus infections. Many viruses associated with chronic
symptomatology in the host will fall in this category.
Infection of T4 cells by HIV can also constitute such a
situation as suggested by Figure 1. HIV replication in T4 cells
was effectively blocked by mismatched dsRNA in tissue culture
(see my published European patent application No. 0,213,921)
despite the fact that HIV replication in these same cells was
only moderately suppressed by IFN alpha, beta or gamma, alone or
in physiological mixtures. Since HIV RNA itself displays
considerable secondary structure which does not block its own
1 336683
expression, different dsRNAs probably elicit different
biological responses. One region of extensive dsRNA structure
at the 5' end of HIV mRNA binds an Mr 15000 polypeptide
suspected to be the HIV transacting (tat) protein (Muesing et
al., Cell, vol. 48, p. 691, 1987) and I suggest that
mismatched dsRNA may compete with HIV RNA for tat protein.
The property of supporting efficient HIV infection, as well as
other chronic viremias, may be a second dsRNA deficiency state
in ARC and AIDS.
EXAMPLE 3 - ~IV Infected Cells
A third kind of dsRNA deficiency state is observed in blood
lymphocytes of individuals with chronic viral infections such as
HIV in ARC and AIDS. Preble et al. (J. Infect. Dis., vol.
152, 1985) reported that these cells possess elevated levels of
dsRNA-dependent 2-5A synthetase, a finding I confirmed. This
result was typical of a series of consecutive ARC and AIDS pa-
tients which I studied and helped form the basis for my inven-
tion in respect of the central role of dsRNA in host recovery
and the necessity to replace dsRNA deficiency conditions by an
appropriate source of exogenous dsRNA.
I reported RNase L to be often depleted in ARC and AIDS
patients (June 6, 1987, The Lancet); however, I reported that
RNase L can return to more normal activity after several weeks
of therapy with mismatched dsRNA in certain cases. As Figure
3B, lane 4 suggests, ARC lymphocyte extracts can inhibit the
activity of normal RNase L under certain conditions. Since
tricholoacetic acid (TCA) and ethanol-soluble extracts lacked
inhibiting activity, the transferable inhibitor observed in ARC
patient's MNC cannot be that described by Williams et al in
herpes-infected cells (Virology, vol. 151, p. 233, 1986).
Also, I consistently saw no inhibitor in MNC extracts from
healthy individuals (Lane 5) studied to date.
Since mismatched dsRNA acts directly on these cells, they
must also be dsRNA-deficient. Thus, I have shown that different
24
1 336683
dsRNAs have different bioactivities (the HIV dsRNA being
inhibitory whereas mismatched dsRNA is not). It appears that
lymphocytes from ARC and AIDS patients are dsRNA-deficient in
the sense that only some of the necessary dsRNA-dependent
functions are carried out. One function at least, the mainte-
nance of sufficient levels of bioactive RNase L, is not carried
out unless exogenous dsRNA is added.
E~MPLE 4 - Chronic Eatigue Sy~ ome
An example of dsRNA deficiency requiring appropriate re-
placement therapy is Chronic Fatigue Syndrome (CFS), a condition
involving some 10 to 12 million Americans, a difficult to
diagnose, ubiquitous disorder characterized by extreme fatigue,
lymph gland enlargement and constitutional symptoms such as
weight loss, loss of appetite and the like. The condition oc-
curs primarily in young, active people. While some CFS patients
manifest neuropsychiatric changes such as depression, loss of
memory and similar derangements, chronic fatigue syndrome is
sometimes difficult to distinguish from entirely neurological
disorders, particularly depression. Various laboratory studies
indicate that many different viruses may replicate in
individuals having Chronic Fatigue Syndrome, and that these
individuals become, in effect, "virus sewers". Viruses such as
Epstein-Barr, cytomegalovirus, retroviruses, herpes viruses,
etc., are often present in such individuals.
The in vivo concentration of 2'-5'A molecules, a
biochemically related, accurate measurement of intracellular
dsRNa levels, in normal individuals and subjects with Chronic
Fatigue Syndrome, is assessed as follows: Ethanol-soluble
fractions of patient samples (Ficoll-Hypaque-purified peripheral
blood lymphocytes) were analyzed for their 2'-5'A content in
2'-5'A core-cellulose assays (affinity chrmoatography (with poly
U-~32P}-pCp. In this assay, the ability of 2'-5'A-activated
RNase L to hydrolyze poly(U) is used to determine the
concentration of functional 2'-5'A.
1 336683
Reference values were established by testing 15 normal
subjects with no recent history of viral infections as evidenced
by lack of fever, absence of constitutional symptoms, rashes,
etc. Concentrations of their lymphocyte 2'-5'A levels were
determined using calibration curves obtained with authentic
2'-5'A molecules. Normal individual reference values, expresses
as nanamoles of 2'-5'A within a range of 0.3 to 1.1 n~n~ole per
gram of PBMC cells.
Using this assay method, ten patients exhibiting the usual
symptoms of Chronic Fatigue Syndrome were tested and the results
obtained were as follows:
TABLE 2
n moles 2'-5'A
Subjectper gram lympl-v~y ~e
Number protein
1 <0.08
2 <0.05
3 <0.05
4 <0.05
nd*
6 nd*
7 <0.01
8 <0.01
<O. 01
<0.08
* not detectable
Patients with Chronic Fatigue Syndrome have generally below
0.1 and always below about 0.2 n moles of 2'-5'A per gram of
lymphocyte protein. Definitive treatment of such individuals
1 336683
with Chronic Fatigue Syndrome is provided by supplying exogenous
dsRNAs, as required, until the intracellular level of 2'-5'A
oligonucleotides reaches normal indicating the return of the
intracellular dsRNA level to normalcy and/or the patient's
clinical symptomology abates. The patient's resistance to
Chronic Fatigue Syndrome and opportunistic viruses is maintained
by continuing to measure the patient's intracellular 2'-5'A
oligonucleotide levels and supplying exogenous dsRNA, as
required, to maintain the 2'-5'A level in the normal range,
usually in excess of 0.2 nanamoles of 2'-5'A per gram of
lymphocyte protein.
The natural dsRNAs play a role in host defense when chal-
lenged with a viral disease such as Chronic Fatigue Syndrome.
Specific reduction in bioactive dsRNA, or enzymes which depend
on dsRNA, notably a viral-associated inhibitor of RNase L
coupled with abnormally low levels of 2'-5'A in peripheral blood
lymphocytes, within specific cells contributes to disease
progression. dsRNA, notably mismatched dsRNAs, reverse disease
progression.
Patients having Chronic Fatigue Syndrome are treated with
intravenous infusions of 200 to 600 mg (depending upon disease
severity and viral burden, etc.) of rI-r(Cl1 14,U) twice
weekly and 1-'5'A levels increase in association with clinical
improvement. The amount of dsRNA administered and the frequency
of administration will be guided by the 2'-5'A levels measured
in conjunction with the patient's clinical improvement. Amounts
of dsRNA administered will provide a level of from 0.01 to 1,000
micrograms of dsRNA per milliliter of the patient's systemic
blood circulation immediately following administration measured
at a point distal from the point of infusion.
EXAMPLE 5 - Antiviral Effect on T4 Cell Population~
dsRNA, particularly mismatched dsRNAs, exogenously admin-
istered restore a viral-infected patient's ability to respond to
viral challenge. As an illustration of a viral condition, HIV
1 336683
was selected as one of the most damaging. The primary target of
an HIV infection are the T4 lymphocytes and their population is
dramatically reduced as the viral infection progresses.
Further, T4 cell population decline is an unwelcomed,
quantifiable disease parameter whose decline reflects a
worsening of the disease. Conversely, an increase in the T4
cell population is a favorable diagnostic indication showing an
improvement in therapeutic intervention.
The normal T4 cell population for a healthy individual is
400 cells per cubic millimeter of blood. Thirty-nine patients
(see Figure 4) diagnosed as being in the pre-ARC or ARC category
were measured and divided into three categories based upon the
absolute number of T4 cells per mm3 of blood: (a) greater
than 300 (18 patients, data not shown); (b) 150 to 300 (13
patients); and (c) less than 150 (8 patients, mean = 92). The
patients in groups (b) and (c) were treated with 100-200 mg
rIn-(C12,U) (Ampligen~) twice weekly by IV infusion for
3 to 16 months (mean = 8.4 months). For purposes of comparison,
T4 cell population data from untreated HIV-infected patients
(181) are included (connected deltas) to show the otherwise
inevitable decline in the T4 population in such individuals.
The baseline is the initial absolute T4 lymphocyte concen-
tration before initiating therapy. Improvement is expressed in
terms of percent change, positive or negative, from this
baseline as shown graphically in Figure 4. Patients were
initially observed and T4 cell concentration measured. The (b)
group of patients, shown in Figure 4 by which the open circle
connected line, had T4 cell concentrations in the range of 150
to 300 cells/mm . These patients received initially lO0 mg
Ampligen (IV twice weekly) as a precautionary measure and
observed for adverse reactions, then the dose was increased to
200 mg. given at the same frequency. A second group of patients
(c) whose T4 cell concentration was less than 150 cells/mm3
were given 300 mg Ampligen (IV, twice weekly). The percent
change from baseline is shown in the line connecting the solid
circles. Untreated patients (181), with absolute T4 lymphocyte
28
1 336683
continued with a sharp decline as shown in the line connecting
the deltas.
Mean T4 cells increased 4.3% after 4 months in 30 patients,
8.0% after 8 months in 10 patients and 17% after 12 months in 6
patients. Only 2 patients (mean baseline T4 = 82 cells)
advanced to AIDS while receiving continuous rIn-(C12,U)n
therapy and another ARC patient who discontinued therapy for 2
months developed Kaposi's sarcoma.
These data demonstrate that infected individuals whose
absolute T4 lymphocyte levels are less than 150 cells/mm
require significant quantities of the drug to slow the rate of
T4 cell reduction; however, patients in the 150-300 range are
effectively maintained at lower dosage levels. These data also
underscore the importance of initiating therapy before drastic
decline in the HIV patient's condition.
These data further demonstrate the direct relationship
between the magnitude of the dsRNA deficit (assessed indirectly
but accurately by absolute T4 cell populations) and the quantity
of exogenous dsRNA required to correct the deficiency and
restore the patient towards full health (as a restoration of the
T4 cell population to values approaching that in less severely
infected and uninfected individuals) -- the larger the T4
deficit, the greater the quantity of dsRNA required to restore
the deficit.
EXAMPLE 6 - d~RNA Replacement Therapy in Paramyxoviru~
Infection
Another example of a transient dsRNA deficiency requiring
appropriate replacement therapy may be various phases in
neonatal life and infant development wherein the body's immune
system is not fully developed. In these instances, potentially
life-threatening infections may emerge from what would otherwise
be self-limiting infections of adolescence or adulthood. A
specific example follows: members of the paramyxovirus family
1 336683
, ~ ~
such as respiratory syncytial virus (RSV) can cause acute
bronchiolitis in infants (typically 6 to 18 months of age) with
their yet-to-be fully developed immune intriasic antiviral
defense systems. In such cases, I show below that replacement
therapy with dsRNA [rIn-r(C12,U)n] can be crucial and
abort what otherwise may be a lethal event (death).
To develop this novel insight, I grew a human amnion-de-
rived cell line designated CCL 25 under standard laboratory
conditions. CCL 25 cells (obtained from the American Type
Culture Collection, Rockville, Maryland, USA) support the growth
and replication of RSV in a manner analogous to human neonatal
bronchiole tissues. The attached table shows conclusively that
I was able to reduce the multiplication of RSV by approximately
99% using doses of dsRNA which I have independently shown are
well-tolerated in humans.
1 336683
TABLE 4
Activity of 2-5A and RNa~e L I~olated from PBMC of
Eumans on ~omopolyribonucleotide~
Source of RNase L: PBMC L929 cells
% Degradationa % Degradationa
Poly(U)- Poly(C) Poly(U)- Poly(C)-
[32P]pCp 32 32 [32P]pCp
Authentic 2-5A 84 0 93 0
2-5A Isolated
from PBMC 34 0 45 0
a average of 3 independent determinations; standard deviation 20%.
Methods. RNase L, purified from extracts (100 ~g pro-
tein) of PBMC from a healthy individual or from L929 cells, were
incubated under core-cellulose assay conditions in the presence
of poly(U)[32P]pCp (11000 dpm, 0.10 ~Ci/mmole) or
poly(C)[32P]pCp (12400 spm, 0.33 ~Ci/nmole) after the addition
of authentic 2-5A (p3A3, 5 x 10 7 M) or 2-5A isolated from
an extract (12.5 ~g protein) of PBMC from patient #5 (at 9 weeks
of mismatched dsRNA therapy). The poly(U)[32P]pCp and
poly(C)[32P]pCp were synthesized from poly(U) or poly(C)
(Sigma) and [32P]pCp (Amersham) by T4 ligase (Bethesda
Research Laboratories). In control experiments (in the absence
of p3A3) 3300 dpm of poly(U)[32P]pCp and 11000 dpm of
poly(C)[ P]pCp were retained on glass fiber filters and are
referred to as no (0%) degradation.
1 336683
Since it is established that infectious center formation
induced by RSV involves direct cell to cell spread of the virus,
I have now established that simply by raising the intracellular
concentration of dsRNA effectively I can reduce the cell to cell
dissemination of RSV without significant untoward effects on
other bodily tissues.
Thus, disease pathology caused by other potentially
self-limited pathogens such as RSV (paramyxovirus family) as
well as other RNA producing viruses which affect the na-
sal/tracheal/bronchial tree, such as rhinoviruses, or influenza
viruses, may be effectively contained by a dsRNA replacement
therapy mechanism. Local portals of pathogen entry, such as
(but not limited to) the airways, may have intrinsically or
transiently lower dsRNA content (dsRNA being herein operation-
ally defined as any of those molecules which trigger the rele-
vant immune/antiviral defense cascades to thwart pathogen
reproduction/tissue-induced pathology).
EXAMPLE 7 - Treatment of ~epatitis
Numerous reviews of liver disease in patients with AIDS
have appeared in the literature over the last 2-3 years. The
recurrent theme is the wide array of histologic findings in
both needle directed biopsy specimens and biopsy at time of
autopsy. In all studies below mentioned the U.S. Center for
Disease Control, Atlanta, Georgia, USA, criteria for a diagnosis
of AIDS was satisfied. Typical presenting symptoms and signs of
patients found to have extensive hepatic disease are entirely
nonspecific and include weight loss, fever, lymphadenopathy,
hepatosplenomegaly, and abdominal pain. Serum transaminases
typically are elevated in most AIDS patients. There is a
propensity for elevated alkaline phosphatases to be
disproportionately high; it has been suggested that such
elevations of alkaline phosphatase are correlated with a
histologic diagnosis of granulomatas disease. Autopsy studies
done in unselected patients (i.e., unbiased patient selection as
1 336683
, . . .
opposed to biopsy studies in patients`with hepatomegaly and
elevated liver function tests) universally demonstrate a wide
array of intrahepatic pathology. Approximately 80-90% of all
autopsy cases in AIDS patients demonstrate abnormal liver
anatomy. Hepatic derangements include vascular congestion,
portal inflammation, steatosis, focal necrosis,
granulomata, bile stasis, cirrhosis and Kupffer cell
hyperplasia (in approximate order of frequency). Of interest,
the intraphepatic diagnosis of Kaposi Sarcoma at autopsy
was the most common specific intrahepatic pathogen (18%) in
one study (Schneiderman et al, Annals Internal Med.,
Vol. 105, pg. 207, 1987).
Biliary disease has recently been described in a small
number of AIDS patients. Isolated case reports have described
sclerosing cholangitis, intrahepatic destructive bile duct
lesions, and benign extrahepatic biliary obstruction in
patients with AIDS. No specific etiologic agent has been
documented in these cases; cytomegalovirus has been implicated
as a possible cause secondary to histopathologic findings on
biopsy. In AIDS patients with benign extrahepatic biliary
obstruction, common presenting complaints have included liver,
right upper quadrant pain and modest bilirubin elevations. At
autopsy or laparatomy a diagnosis of ampulai of vater
structure secondary to fibrosis was made. In at least two
cases of extrahepatic obstruction, cryptosporidia were
isolated from the biliary tree. Other cases were notable for
cytomegalovirus inclusive bodies in the vicinity of the
ampulae of vater. As with the above cases of intrahepatic
cholangitis, specific etiology is unknown and the specific
pathogens may have only a casual role in the pathogenesis of
these disorders.
I determined that dsRNA infusion [rI-r(C12,U)n, 200
mg, twice weekly, IV] also corrects or significantly
ameliorates the underlying cellular abnormality associated with
hepatitis B virus (HVB) multiplication. To accomplish this
insight, the following method was used:
33
1 336683
Evaluation of Sera or Plasma for HBV-Specific DNA: The presence
of serum (or plasma)-associated HBV DNA, an indicator of the
presence of hepatitis B virus, was determined by slot-blot
hybridization. Serum samples were diluted in NH40Ac to a
final concentration of 1 M salt. Using a 72-wall
microfiltration apparatus (Schleicher & Schuell, Keene,
N.H.), the samples were applied directly to a nitrocellulose
filter that had been presoaked for 10 minutes in 1 M
NH40Ac. The DNA was denatured in situ with alkali
and then neutralized. Dilutions of purified HBV DNA in
normal human serum werè assayed in an identical manner to
quantitate each slot-blot hybridization. The filters were
baked, prehybridized, hybridized, and washed under conditions
of high stringency, and autoradiographed using standard
conditions (Maniatis, T., Fritsch, E.F. and Sambrook, J.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laoratory, Cold Sprig Harbor, 1982). The HBV probe DNA used
in the hybridizations was obtained from a recombinant
plasmid containing a single, unit length copy of HBV DNA
(HBsAG serotype adw2) cloned at the EcoRl site. The
plasmid was digested with EcoRl and the HBV DNA was
purified by 2 cycles of preparative agarose gel
elecrophoresis and electroelution. The purified HBV DNA
was labeled with 32P-dCTP to a specific activity of 1-2 x
109 cpm/~g using the random-prime labeling technique
(Feinberg, A.P. and Vogelstein, B., Analyt. Biochem.,
132:6-13, 1983). The level of sensitivity obtained with this
assay is 1 pg of HBV DNA. At this level of sensitivity, I
am detecting 2.9 x 105 molecules of HBV DNA.
The autoradiographs produced were eval~ated for degree of
relative intensity and numerically ranked according to observed
intensity and are reported in the following Table. Days of
therapy are given parenthetically.
34
1 336683
Table 4
Effect of dsRNA Administration on Reducing Concentration
of HBV (hepatitis B virus) DNA in Bodily Fluid
Patient #1 (day sampled) Patient #2 (day sa~pled) Patient #3 (day sa~pled)
SaDple Nu~ber Blot Intensity P Therapy Blot Intensity Blot Intensity
T 3+ (0) 3+ (0) 3+ (0)
1 3+ (20) 3+ (21) 3+ (31)
2 3+ (64) 2.5+ ~42) 3+ (72)
3 3+ (106) 2+ (56) 3+ (86)
4 2+ (162) 2+ (84) 3+ (115)
2+ (219) 1.5+ (140) 2+ (170)
6 2+ (266) 1 (180) 2+ (227)
7 1.5+ (310) 1.5+ (270)
8 1+ (353) 1.5+ (296)
9 1+ (400) 1.5+ (324)
1+ (450) 1.5+ (387)
Legend: T = zero (pre-therapy). refers to
relative intensity of x-ray film exposed for five days to 32p
containing viral DNA. Larger pluses (e.g., 3 vs. 1 )
refer to more intense and larger slot-blots on x-ray film
indicating presence of more viral DNA. In order to increase
sensitivity of viral measurements, various dilutions of patient
sera were used, typically dilutions of 1:40, 1:200, 1:40 and
1:1,500. Confirmatory results were established with
densitometric tracings of slot blots. Methodology was based on
reagents provided by Schleicher and Schnell (materials
designated No. 370 2/284). Complementary results were obtained
with analysis of viral specific antigen (immunoassay for the
detection of hepatitis B surface antigen in human serum or
plasma) kit available from Abbott Laboratories (designated
technical exhibit 83-0804/R12 dated July 1985)
As the results of my invention become known publicly and
1 336683
further developed and applied to additional conditions/infec-
tions/disorders, it may well become possible to make a clinical
diagnosis of dsRNA deficiency without relying on extensive labo-
ratory date and perhaps even the scientific insight and novelty
I put forth in this document. Many less obvious
dsRNA-deficiency states can also exist. For example, since
IFN operates naturally as a feback inhibitor of
proliferative pathways (Aullo et al, Cell, Vol. 43, pp.
793, 1985), and since dsRNA-dependent mediators implement these
events, then cells undergoing differrentiations or with
tumorigenic/metastaic potential may be dsRNA-deficient.
From the examples cited above, it is apparent that by
interrelating biologic function (e.g., cell response to
exogenous IFN) or biochemical function (e.g., elevated 2-5A
synthetase) or presence of 2'-5' oligoadenylate pathway
intermediates, I can readily diagnose dsRNA deficiency states
and quantitate the degree of derangement by various means
including:
(a) Determination of endogenous 2',5' oligoadenylate
concentrations.
(b) Quantitative analysis of 2-5A concentration and molecu-
lar size of 2-5A from patient samples via high pres-
sure liquid chromatography.
(c) Proof of biological functionality of the 2-5A synthe-
sized in vivo in patients utilizing ribosomal RNA
cleavage assays.
(d) Binding assays with 2', 5'-p3A4{32P}-3'-pCp to
determine the level of free (unbound) RNase L in pa-
tients' samples.
(e) Core-cellulose assays (affinity chromatography) with
poly U-{32P}-pCp to characterize the specificity of
activation of RNase L by 2-5A synthesized in patient
samples.
(f) Ribosomal RNA cleavage assays to determine the level
of activate RNase L by 2-5A synthesized in patient
samples.
36
- 1 336683
(f) Ribosomal RNA cleavage assays to determine the level
of activated RNase L in patients samples.
Some of these methods are described in detail in my
copending Canadian patent application entitled "Double Stranded
RNA Correction of Aberrant Metabolic Pathways", Serial No.
572,123 filed July 15, 1988.
DsRNA sufficiency ~tates can also exist. For example,
CML and hairy cell leukemia (HCL) are often responsive to IFN
given as sole therapy and reflect intracellular situations of
natural dsRNA sufficiency. To support this notion, I found
similar levels of 2-5A synthetase-activating dsRNA in nuclei of
mononuclear blood cells form untreated patients with HCL as in
nuclei of IFN-treated Hela cells.
Mismatche~ d~RNA. While many dsRNAs can induce various
lymphokines, including IFN, and activate 2-5A synthetase, not
all dsRNAs have these properties. Of synthetic dsRNAs,
poly(I):poly(C) is an excellent inducer of IFN and activator of
2-5A synthetase but is also very toxic. Poly(I):poly(C12,U),
also referred to as "mismatched dsRNA" or Ampligen0 (HEM Re-
search, Inc., Rockville, Maryland, USA), contains periodic
uracil residues in the polypyrimidine strand. Mismatching induc-
es rapid biodegradation without destroying biological function.
For example, poly(I):poly(C12,U) exhibits antiviral, antitumor
and immune-enhancing activity, and is nontoxic.
DsRNA As A Replacement Therapy in Broad Spectrum Viral and
Cancer TheraPy. Mismatched dsRNA may be particularly well-suit-
ed for chronic viral infections including ARC treatment because
it is both a broad-spectrum antiviral and an immune enhancer;
even at high doses it is essentially without long term toxici-
ty. My data on 45 consecutive patients treated intraveneously
with 200-250 mg of poly(I):poly(C12,U) twice weekly showed
rapid decline in virus concentration (HIV, CMV and herpes) and
durable enhancement of both T and 8 cell immunity accompanied by
,~
.~ .
1 336683
improved performance with no significant ~ide effects. In many
patients, the clinical responses continue over approximately 18
months, or as long as they continued dsRNA therapy (100-400 mg
twice weekly). In so~e cases larger doses, more frequently
applied, may be necessary. Viral infections and immune lesions
in AIDS patients may differ from one individual to another and
no single agent can be expected to reverse all the potential
clinical problems. However, dsRNA may play a pivotal role in
various treatment regimens. For example, since mismatched dsRNA
displayed synergy with AZT in blocking ~IV infection of T4 cells,
mismatched
asKNA an~ very low dose AZT may be an important combination in
early phases of infection including ARC. Both dsRNA and IL-2
enhance T cell, NK cell and LAK cell activity and thus far,
where combined, I have shown that the two biologicals have dis-
played therapeutic synergism without added toxicity. Further, I
anticipate that, insofar as IFNs enhance differentiation of B
cells and monocytes, certain lymphokines may increase the abili-
ty of dsRNA to promote ~pecific neutralizing antibodies against
HIV and increase monocyte killing activity. Similarly, since
retinoids increase the number of T4 cells and promote differenti-
ation of both T and B cells with IFN, the combination of dsRNA
and retinoids may provide additional benefits in ARC and AIDS.
The combinations of dsRNA and thymic peptides may also have T4
cell restorative effects, especially in patients with very low
T4 cell numbers. Finally, the fact that dsRNA exhibits direct
antiproliferative activity against Kaposi'~ ~arcoma cells which
can be augmented by adding either IFN alpha or IL-2, suggests a
new antitumor regimen in advanced disease. Control of blood
cell parameters in AIDS will reveal increased CNS problems. To
that end, mismatched dsRNA crosses the murine blood brain barri-
er in sufficient quantities to activate oligo 2'5' adenylate
synthetase in brain parenchymal cells; thus, combinationæ of
dsRNA and other agents in~luding lymphokines which cross the
blood-brain barrier will be useful in viral-related dementias.
Lymphokines will be understood to include the interferons, pref-
- 1 336683
erably interferon alpha, the interleukins, specifically
interleukin-2 (IL-2) and recombinant interleukin-2 (rIL-2), and
tumor necrosis factor (TNF). Also included are lymphokine acti-
vated killer (LAK) cells formed in animals in response to expo-
sure to a lymphokine.
When interferon (alpha) is used as the lymphokine, an
amount of from 0.01 to 100,000 IRU per milliliter of the pa-
tient's body fluid is provided. When IL-2, preferably rIL-2, is
the lymphokine, the amount administered lies within a range of
about 102 IL-2 units per kg of the patient's body weight up to
a value approaching unacceptable levels of toxicity in the pa-
tient, which may be as high as 106 IL-2 units. However, most
effective, toxic-reaction manageable values are in the range of
from about 103 to about 104 IL-2 per kg of body weight.
The usual amounts of dsRNA administered provide a level of
from 0.1 to 1,000 micrograms dsRNA per milliliter of the pa-
tient's body fluid. The term body fluid is intended to refer to
that solution of serum, salts, vitamins, etc., which circulates
within the organism and bathes the tissues. When both agents (a
dsRNA and a lymphokine) are administered they may be adminis-
tered as a mixture, administered separately but simultaneously,
or sequentially.
Administration of a dsRNA and a lymphokine "in combination"
includes presentations in which both agents are administered
together as a therapeutic mixture, and also procedures in which
the two agents are administered separately but simultaneously.
FIGURE 1 is a flow chart illustrating a typical dsRNA defi-
ciency which leads to host morbidity and dsRNA sufficiency which
leads to host recovery. Bioactive dsRNA is produced
intracellularly or introduced by certain viruses (e.g., EMC) and
triggers a series of enzymatic events, including lymphokine
production and mediator activation, which lead to an antiviral
state as well as immune cell differentiation and host recovery.
DsRNA introduced by various viruses (e.g., HIV) or tumor cells
can subvert this pathway by acting as an inhibitor. Physiologi-
39
1 3366~3
cal events can limit dsRNA production or cause production ofaberrant dsRNA. Such abnormalities lead to chronic infections
and hose morbidity. In ~IV and other chronic viral infections,
immune dysfunction can also result from an inhibitor of RNase L
(shown as an alternate pathway) which is overcome by a Eupply of
appropriately configured exogenous dsRNA.
FIGURE 2A and 2B illustrate induction of 2'5'
oligoadenylate synthetase by mismatched dsRNA in MNC or a CML
patient. A CML patient was treated with IFN alone for seven
days (Fig. 2A), then with mismatched dsRNA and IFN (Fig. 2B).
At times before and during treatment, synthetase activity was
measured in Ficoll-purified MNC.
FIGURE 3A is a photograph of polyacrylamide gel
electrophoresis plates showing the indicate number of tracks and
bands or zones along each track. Elevated activity of RNase L
associated with novel cleavage products in MNC of a CML patient
are illustrated on this plate. Hungarian L929 cell~ which
supply rRNA but no RNase L, are depo6ited under the terms of the
Budapest Convention at the American Type Culture Collection as
ATCC No. CRL 9659; L929 cells as deposited as CCL 1 and
MNC from the CML patient were prepared according to Kariko and
Ludwig and Silverman et al, then RNase L from the MNC was
measured as described in my copending Canadian application
Serial No. 572,123 filed July 15, 1988.
FIGURE 3B is also a photograph of a polyacrylamide gel
electrophoresis plates showing the presence of an ~Nase L
inhibitory factor in PBMC extracts of an ARC patient as deter-
mined in the rRNA cleavage a6say. Extracts of L929 cell6 (which
supply both RNase L and rRNA) and MNC were prepared according to
Kariko and Ludwig and Silverman et al. respectively.
Lane 1: 18 ~1 L929 cell extract (1~0 ~g total protein) was
1 336683
incubated in the presence of 4 ~1 NP40 buffer used for MNC lysis
plus 5.5 ~1 water.
Lane 2: identical to Lane 1, except that in lieu of water, 5.5
~l of 5 x 10 8 M authentic P3A3 was added to incubations.
Lane 3: identical to Lane 1 except that 5.5 ~l of TCA-soluble
extract from MNC extract (100 ~g total protein) from an ARC
patient was added prior to incubation.
Lane 4: 18 ~1 L929 cell extract was incubated with 4 ~l MNC
extract from an ARC patient (100 ~g total protein) and 5.5 ~1
water.
Lane 5: 18 ~l L929 cell extract was incubated with 4 ~l MNC
extract from a healthy individual (100 ~g total protein) and 5.5
~l water. 28S and 18S rRNA as well as normal levels of 2'5'A
activated RNase L specific cleavage products are indicated.
41
