Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
93/230~7 PCT/US93/045i~
1~
,,.
DESCRIPTION ~
METHOD AND REAGENT FOR INHIBITING -
CANCER DEVELOPMENT
Backaround of the Invention
This invention relates to methods for treating
cancer, and in particular, growth of a transformed cell,
and inhibition of progression to a transformed phenotype
; 5 in pre-neoplastic cells.
Transformation is a cumulative process whereby
~` normal control of cell growth and differentiation is
interrupted, usually through the accumulation of mutations
affecting the expression of genes that regulate cell
growth and differentiation.
Scanlon WO91/~18625, WO91/18624, and W091~18913
describes~;a~ rlbozyme effe~ctive~to cleave oncogene RNA in
~ the~ H-ras gene.~ This ribozyme is said to inhibit C-fos
:~ expressi~on ln response to cis-plantin or other stimuli.
15~ ~Reddy;,~WO 92/OOQ80 and U.S. Serial No. 07/544,199 (filed
3une~26,~19~90~, describes use of ribozymes as therapeutic
agents~or 1eukemla~s, such as chronic myelogenous leukemia
CML)~ by~targeting specific junction regions of the
bcr-abl ~ f~usion transcrlpt.
- SummarY of the Invention
This inventlon concerns use of a ribozyme
t~argeted~to ~the P-glycoprotein (mdr-1 gene) or other
cancer-related genes prior to and/or during administration
of~anticancer chemotherap~u!tilc agents. Inclusion of such -~
a ribozyme increases the susceptibility of the transformed
cells to such agents.
Applicant notes that relapse of disease caused :`,
by cancerous ~ cells after administration of
chematherapeutic agents is a major problem in obtaining
lasting remissions in a clinic. In some neoplasias,
relapse is caused by the expansion of a population of
transformed cells resistant to the initial and subsequen~
-,~; ~ . .-.
WO93/230~7 ~ 4 ~ 9 PCT/US93/0~
forms of chemotherapy due to inappropriate expression of
the mdr-1 gene, also called P-glycoprotein. Such
expression is usually caused by selection of transformed
cells that have amplified the mdr-1 gene and thus produce
increased amounts of the mdr-1 gene product. Applicant
describes treatment of and prevention of this condition by
use of ribozymes targeted to the mRNA encoded by this
gene.
The mdr-1 gene encodes a 170 kDa integral
membrane transport protein that confers resistance to
certain chemotherapeutic agents such as colchicine,
doxorubicin, actinomycin D and vinblastine ~reviewed ln
Gottesman and Pastan, 263 J. Biol. Chem. 12163, 1988).
The gene has been isolated from both human and rodent
cells selected in vitro for resistance to such agents
(Roninson et al., 309 Nature 626, 1984; and Roninson et
al., 83 Proc. Natl. Acad. Sci USA, 4538, 1986), and the
entire;4.5-kb MDRl transcript encoding the human MDRl has
been sequenced (Chen et al., 47 Cell 381, 1986, EMBL
accession # M14758). The gene is normally expressed in
the cells of the colon, small intestine, kidney, liver and
adrena~l gland. High levels of MDR1 transcript have been
found in adenocarcinomas that are intrinsically resistant
to a broad range of chemotherapeutic agent~, such as those
derived from adrenal, kidney, liver and bowel.
The in~ention features use of ribozymes to
-~ inhibit the development or expression of a transformed
, ~ .
phenotype in man and other animals by modulating
e~pression of a gene that either contributes to, or
inhibits ~he expression of CML, promyelocytic leukemia,
Burkitt's lymphoma, acute lymphocytic leukemia, follicular
lymphoma, B-cell acute lymphocytic leukemia, breast
cancer, colon carcinoma, neuroblastoma, lung cancer, and
other neoplastic conditions. Cleavage of targeted mRNAs
expressed in pre-neoplastic and transformed cells elicits
-~ inhibition of the transformed state.
.~,;
~ 93/23057 2 1 ~ 5 ~1 ~ 9 PCT/US93/0457~
Riboz~mes are RNA molecules having an enzymatic
activity which is able to repeatedly cleave other separate
RNA molecules in a nucleotide base sequence specific
manner. Such enzymatic RNA molecules can be targeted to
virtually any RNA transcript and efficient cleavage has
been achieved in vitro. Kim et al., 84 Proc. Natl. Acad.
Sci. USA 8788, 1987; Haseloff and Gerlach, 334 Nature 585,
1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17
Nucleic Acids Research 1371, 1989.
Ribozymes act by first binding to a target RNA.
Such binding occurs through the target RNA binding portion
of a ribozyme which is held in close proximity to an
enzymatic portion of the RNA which acts to cleave the
target RNA. Thus, the ribozyme first recognizes and then
binds a target RNA through complementary base-pairing, and
once bound to the correct site, acts enzymatically to cut
the target RNA. Strat~egic-cleavage of such a target RNA
will destroy its ability to direct synthesis of an encoded
protein. After a ribozyme has bound and cleaved its RNA
20~target lt is released from that RNA to search for another
~-~ target~ and can repeatedly bind and cleave new targets.
The enzymatic nature of a ribozyme is
advantageous over other technologies, such as antisense
~technology twhere a nucleic acid molecule simply binds to
--25 a nucleic acid target to block its translation) since the
effective concentration of ribozyme ne`cessary to effect a
therapeutic treatment is lower than that of an antisense
oligonucleotide. This advantage reflects the ability of
the ribozyme to act enzymatically. Thus, a single
~ "
ribozyme molecule is able to cleave many ~molecules of
target RNA. In addition, the ribozyme is a highly
specific inhibitor, with the specificity of inhibition
depending not only on the base pairing mechanism of
binding, but also on the mechanism by which the molecule
inhibits the expression of the RNA to which it binds.
That is, the inhibition is caused by cleavage of the RNA
target and so specificity is defined as the ratio of the
:
.~ 1 3 .i ~ 1t.~
W093/230~7 PCT/US93/045~
i i
rate of cleavage of the targeted RNA over the rate of
cleavage of non-targeted RNA. This cleavage mechanism is
dependent upon factors additional to those involved in
base pairing. Thus, it is thought that the specificity of
action of a ribozyme is greater than that of antisense
oligonucleotide binding the same RNA site. ~-
This class of chemicals exhibits a high degree
of specificity for clea~age of the intended target mRNA.
Consequently, the ribozyme agent will only affect cells
expressing that particular gene, and will not be toxic to
normal tissues.
Thus, the invention features novel enzymatic RNA
molecules, or ribozymes, and methods for their use for
inhibiting cancer-related mRNA expression. Such ribozymes
can be used in a method for treatment of disease caused by
expression of the cancer-related genes in man and other
animals, including other primates. This conclusion, as
noted above,~ is based upon the finding that many forms of
cancer~become~ unresponsive to certain chemotherapeutic
agents~as a result of overexpression of, e.a., the mdr-1
gene. The advantage of using ribozymes of the present
invention is their ability to specifically cleave the
:~ ,;
targeted mRNA, ultimately leading to a reduction in target
gene ~activity throu~h a decrease in level of the gene
2~5~ product~. Use of mdr-1 specific ribozymes removes the
--- mechanism of drug resistance used by transformed cells,
and thus enhances drug therapies for tumor cell growth.
These agents can be administered prior to and during
chemotherapeutic treatment of those neoplasias known to
have a high incidenc'e of drug resistance, or can be used
prophvlactically for all neoplasias.
The invention can also be used to treat cancer
or pre-neoplastic conditions. Two preferred
- administration protocols can be used, either i~ vivo
administration to reduce the tumor burden, or ex vivo
treatment to eradicate transformed cells from tissues such
as bone marrow prior to reimplantation.
~3/23057 ~ t~ ~ 9 PCT/U~93/04573
. .
,
Thus, in a first aspec~, the invention features
an enzymatic RNA molecule (or ribozyme) which cleaves mdr-
1 mRNA (i.e., mRNA expressed from the mdr-1 gene), or its
equivalent. In particular, the invention features
hammerhead ribozymes designed to cleave accessible areas
of the mdr-1 mRNA. Such areas include tr.~se sequences
shown in Fig. 2.
In a second aspect, the invention features an
enzymatic RNA molecule (or ribozyme) which cleaves mRNA
associated with development or maintenance of CML,
promyelocytic leukemia, surkitt~s lymphoma or acute
lymphocytic leukemia, follicular lymphoma, s-cell acute
lymphocytic leukemia, breast cancer, colon carcinoma,
neuroblastoma, and lung cancer, including mRNA targets
disclosed in Figs. 3 to 11. Such mRNA is recognized by
those in the art to encode an aberrant cel.ular protein
which is able to control cellular proliferation, and is
directly linked to (correlated with) the presence of the
leukemic phenotype.
20 ~ By "enzymatic RNA molecule~ it is meant an RNA
olecule which has complementarity in a substrate binding
-, :
region to a specified mRNA target, and also has an
~ enzymatic activity which is active to specifically cleave
-~ RNA in that mRNA. That is, the enzymatic RNA molecule is
able to intermolecularly cleave mRNA and thereby
inactivate a target~mRNA molecule. This complementarity
functions to allow sufficient hybridization of the
~,
enzymatic RNA molecule to the target RNA to allow the
cleavage to occur. For in ~ivo use, such complementarity
may be be'tweén `30 and '45 bases. One hundred percent
-complementarity is preferred, but complementarity as low
as 50-75% may also be useful in this invention.
: By 11 equivalentll RNA to mdr-1 mRNA is meant to
include those naturally occurring mRNA molecules
associa'ced with neoplastic diseases in various animals,
including humans, and other primates, which have similar
~ ~ .
~ - structures and functions to that mdr-1 mRNA in humans.
~ J ~
w0~3/230~7 PCT/US93/0457~: -
The deduced sequences of the mouse and human P-
glycoproteins are 80~ identical.
In preferred embodiments, the e~zymatic RNA
molecule is formed in a hammerhead motif, but may also be
formed in the motif of a hairpin, hepatitis delta virus,
group I intron or RNaseP-like RNA (in association with an
RNA guide sequence). Examples of such hammerhead motifs
are described by Rossi et al., 8 Aids Research and Human
Retroviruses 183, 19~2; of hairpin motifs by Hampel et
al., ~RNA Catalyst for Cleaving Specific RNA Sequences'~,
filed September 20, 1989, which is a continuation-in-part
of U.S. Serial No. 07t247,100 filed September 20, 1988,
Hampel and Tritz, 28 Biochemistrv 4929, 1989 and Hampel et
al., 18 Nucleic Acids Research 299, 1990; an example of
the hepatitis delta virus motif is described by Perrotta
and Been, 31 B1ochemistrY 16, 1992; of the RNa~seP motif by
Guerrier-Takada ~et al.~, 35 Cell 849,~ 1983; and of the
group~ ntron;by Cech et al., U.S. Patent 4,987,071.
These~specif1c motlfs are not limiting ln the invention
20~-a~d~those ~skilled~ n the art will recognize that all that
is~ lmportant ln an enzymatic RNA molecule of this
invention is that it has a specific substrate binding site
which is~ complementary to~one or more of the target mRNA
regi~ons,~ and that it have nucleotide sequences within or
25~ surrounding that substrate binding site which impart an
mRNA~cleaving activity~to the molecule.
In a related aspect, the invention features a
: . ,. - . - ,
mammalian cell which lncludes an enzymatic RNA molecule as
described above. Preferably, the mammalian cell is a
human or other primate cell.
In another related aspect, the in~ention
features an e~pression vector which includes nucleic acid
encoding the enzymatic RMA molecules described above,
located~in the vector, ~ , in a manner which allows
expression of that enzymatic RNA molecule within a
mammalian cell.
~"
.) 93t230~7 r~ 1 3, ~ q 9 Pcr/ US93/0457 t~
In yet another related aspect, the invention
feature~ a method for treatment of an mdr-l gene-related
disease, chronic myelogenous leukemia (CML), promyelocytic
leukemia, surkitt~s lymphoma or acute lymphocytic
leukemia, follicular lymphoma, B-cell acute lymphocytic
leukemia, breast cancer, colon carcinoma, neuroblastoma,
or lung cancer, by administering to a patient an enzymatic
RNA molecule as described above.
In another related aspect, the invention
features a method for treatment of CML by ex vivo
treatment of blood or marrow cells with an enzymatic RNA
molecule as described above.
The invention provides a class of chemical
cleaving agents which exhibit a high degree of specificity
for ~he mRNA causative of CML, promyel~cytic leukemia,
Burkitt's ~lymphoma or acute lymphocytic leukemia,
follicular lymphoma, B-cell acute lymphocytic leukemia,
~ breas~ cancer, colon ~carcinoma, and neuroblastoma. If
- desired, such~ ribozymes can be designed to target
2~0~ equ~1valent~ slngle-stranded DNAs by methods known in the
art.~The~ribozyme molecule is preferably targeted to a
highly cQnserved sequence region of the mdr-1 mRNA. Such
enzymatic RNA molecules can be delivered exogenously to
affect~ed~cells or endogenously to infected cells. In the
25 ~preferred hammerhead~ motif~the small size (less than 40
nucleotides, preferably between 32 and 36 nucleotides in
length) of~the molecule allows the cost of treatment to be
reduced compared to other ribozyme motifs.
-~ The smallest ribozyme delivered for any type of
treatment`reported to date (by Rossi et al.,'1992, supra)
-is an in vitro transcript having a length of 142
-~ nucleotides. Synthesis of ribozymes greater than 100
- nucleotides in length is very difficult using automated
methods~ and the therapeutic cost of such molecules is
prohibitive. Delivery of ribozymes by expression vectors
is primarily feasible using only ex vivo treatments. This
limits the utility of this approach. In this invention,
w093t230~7 PCT/US93/04
small ribozyme motifs ~e.q., of the hammerhead structure,
shown generally in Fig. 1) are used for exogenous
delivery. The simple structure of these molecules also
increases the ability of the ribozyme to invade targeted
S regions of the mRNA structure. Thus, unlike the situation
when the hammerhead structure i5 included within longer
transcripts, there are no non-ribozyme flanking sequences
to interfere with correct folding of the ribozyme
structure, as well as complementary binding of the
ribozyme to the mRNA target.
The enzymatic RNA molecules of this invention
can be used to treat human CML, promyelocytic leukemia,
Burkitt's lymphoma, acute lymphocytic leukemia, follicular
lymphcma, B-cell acute lymphocytic leukemia, breast
cancer, or lung cancer. Affected animals can be treated
at the time of cancer, or in a prophylactic manner. This
-~ timing of treatment wi~l~l reduce the number of affected
cells and disable cellular replication. This is possible
because the ribozymes are designed to disable those
20~ structures required for successful cellular proliferation.
Ribozymes of this invention may be used as
diagnostic tools to examine genetic drift and mutations
within diseased cells. The close relationship between
rlbozyme activity and the structure of the target RNA
allows the detection of mutations in any region of the
molecule which ~alters the base-pairing and three-
dimensional ~structure of the target RNA. By using
multiple ribozymes described in this invention, one may
map nucleotide changes which are important to RNA
structu~re and functioh in vitro, as well as in cells and
tissues. Cleavage of target RNAs with ribozymes may be
used to inhibit gene expression and define the role
(essentially) of specified gene products in the
progression of disease. In this manner, other genetic
targets may be defined as important mediators of the
disease. These experiments will lead to better treatment
~- ~ of the disease progression by affording the possibility of
.
,
~93/23057 ~ 3 1 ~ ~ 3 PCT/US93/045
combinational therapies (e.q., multiple ribozymes targeted
to different genes, ribozymes coupled with known small
molecule inhibitors, or intermittent treatment with
combinations of ribozymes and/or other chemical or
biological molecules).
Other features and advantages of the invention
will be apparent from the following description of the
preferred embodiments thereof, and from the claims.
Descri~tion of the Preferred Embodiments
-The drawings will first briefly be described.
Drawinas
Fig. 1 is a diagrammatic representation of a
hammerhead motif ribozyme showing stems I, II and III
(marked (I), (II) and (III) respectively) interacting with
lS a target region. The 5' and 3~ ends of both ribozyme and
targ~et are shown. Dashes indicate base-paired
nucleot~ides.
Figs. 2 ~- 11 are preferred targets for mdr-l
gene, chronic myelogenous leukemia, promyelocytic
leukemia, ~Burkitt~s lymphoma or acute lymphocytic
- leukemia~,~follicular lymphoma, B-cell acute lymphocytic
leukemla~, breast cancer, colon carcinoma, neuroblastoma,
and l-ung cancer, respectively.
; Tarqet Sites~
2S ~ ~ Ribozymes targeting selected regions of mRNA
associ~ated with tumor cell growth are preferably chosen to
cleave the target RNA in a manner which inhibits
~translation of the mRNA. Genes are selected such that
inhibition of translation will preferably inhibit cell
~- 30 replication, e.a., by inhibiting production of a necessary
~- protein. Selection of effective target sites within these
critical regions of mRNA entails testing the accessibility
, ~, ~.,
of the target mRNA to hybridization with various
oligonucleotide probes. These studies can be performed
35~ using~ RNA or DNA probes and assaying accessibility by
cleaving the hybrid molecule with RNaseH (see below).
Alternatively, such a study can use ribozyme probes
j 1 3 r~ 3 ~ -
W~93/23057 PCT/US93/04
designed from secondary structure predictions of the
mRNAs, and assaying cleavage produc~s by polyac~ylamide
gel electrophoresis (PAGE), to detect the presence of
cleaved and uncleaved molecules.
The following are examples of cancer conditions
which can be targetted in this manner.
Chronic MYeloqenous Leukemia
Chronic myelogenous leukemia exhibits a
characteristic disease course, presenting initially as a
chronic granulocytic hyperplasia, and invariably evolving
into an acute leukemia which is caused by the clonal
expansion of a cell with a less differentiated phenotype
(i.e., the blast crisis stage of the disease). CML is an
unstable disease which ultimately progresses to a terminal
stage which resembles acute leukemia. This lethal disease
~ affects approximately 16,000 patients a year.
;~ Chemotherapeutic a~gents such as hydroxyurea or busulfan
~ can reduce the leukemic burden but do not impact the-life
. ~
expectancy~of the patient (e.q., approximateiy 4 years).
Consequently, CML patients are candidates for bone marrow
- transplantation (BMT) therapy. However, for those
patients which survive BMT, disease recurrence remains a
major obstacle. Apperley et al., 69 Br. J. Haematol. 239,
1988.
~25 ~ The Philadelphia (Ph) chromosome which results
; ~ ~ from the translocation of the abl oncogene from chromosome
-~9 to the bcr gene on chromosome 22 is found in greater
than 95% of CML patients and in 10-25% of all cases of
acute lymphoblast1c leukemia (ALL). Fourth International
Workshop on Chromosomes in Leukemia. 11 Cancer Genet.
CYtoqenet. 316, 1982. In virtually all Ph-positive CMLs
and approximately 50% of the Ph-positive ALLs, the
leukemic cells express bcr-abl fusion mRNAs in which exon
2 (b2a2 junction) or exon 3 (b3a2 junction) from the major
` 35 breakpoint cluster region of the bcr gene is spliced to
exon 2 of the abl gene. Heisterkamp et al., 315 Nature
758, 1985, Shtivelman et al., 69 Blood 971, 1987. In the
. ~
-
i~93/23057 ~ PCT/US93/04~7~
~.
11
remaining cases of Ph-positive ALL, the first exon of the
bcr gene is spliced to exon 2 of the abl gene. Hooberman
et al., 86 Proc. Natl. Acad. Sci. USA 4259, 1989,
HeisterXamp et al., 16 Nucleic Acids Research 10069, 1988.
The b3a2 and b2a2 fusion mRNAs encode 210 kd
bcr-abl fusion proteins which exhibit oncogenic activity.
Daley et al., 247 Science 824, 1990, Heisterkamp et al.,
344 Nature 251, 1990. The importance of the bcr-abl
fusion protein Ip210bCr-abl) in the evolution and maintenance
10 of the leukemic phenotype in human disease has been -
demonstrated using antisense oligonucleotide inhibition of
p210bC~-abl expression. These inhibitory molecules have been
shown to inhibit the in vitro proliferation of leukemic `-
cells in bone marrow from CML patients. Szczylik et al.,
253 Science 562, 1991. -
- c-MYc Gene~
;c-Myc, when activated, can lnduce malignancy in
~ a~;var~i-et`y o~f tissues, most notably hematopoietic tissues
?,`,'~ (Leder~et al.,~222 Sclence 765, 1983). The~most common
mechanl~sm of; c-myc activation is translocation to any of
- the immunoglobulin (Ig) or T-cell receptor~loci during
lymphoid maturation (Croce and Nowe11, 65 Blood 1, 1985;
Kl;ein ~and~ Klein, 6 Immunol. Today 208, 1985). For
example,~ ln Burkitt's lymphoma the c-myc locus on
chromosome 8 translocates most often to the Ig heavy chain
locus on chromosome 14, but also to the lambda or kappa
light chain Ig genes on chromosomes 2 and 22 (Magrath, in
~; ~ "Epstein-Barr Virus and Associated Diseases", M. Nijhoff
Publishing:631l j1,986).~ , In some instances the c-myc
transcription region is altered in the non-coding exon 1
region; in such cases transcription is initiated at a
cryptic promoter present in the first intron of the c-myc
locus. These rearrangements are thought to lead to
- deregulation of c-muc expression.
c-Myc is not normally expressed in quiescent
cells, but is temporally expressed in actively-dividing
,
~ 1 r;3 5 4 ~
W093/230~7 PCT/US93/04
12
cells, most prominently during transition from Go to G1
phases of growth induction.
Experiments with transfected cell lines and
transgenic animals have shown that c-myc activation plays
a critical role, but is not sufficient for transformation
(Adams et al., 318 Nature 533, 1985; Lombardi et al., 49
Cell 161, 1987; Schwartz et al., 6 Mol. Cell. Biol. 3221,
1986; Langdon et al., 47 Cell 11, 1986). Targeted
inhibition of c-myc expression in tumor cell lines using
antisense oligonucleotides has shown that c-myc expression
is required for growth in certain lymphomas (McManaway et
al., 335 Lancet 808, 1990).
Bcl-2 Gene
The bc1-2 gene is abnormally expressed in about
15~ 85% of follicular lymphomas and about 20% of diffuse
lymphomas due to a t(14,18)(q32;q21) ~ chromosomal
rearrangement~between the bc1-2 locus on chromosome 18 and
the immunoglobulin heavy chain locus on chromosome 14
(Yunis et al.,~ 316 N. Ena1. J. Med. 79, 1987). This
20;:chromosoma1~rearrangement represents the most common found
in~-ly ~ id malignancies in humans~ A bc7-2/IgH fusion
mes~sag~e~is expressed; however, the bc1-2 protein-coding
~-~ region is not interrupted since the major breakpoint
region~lies~ ln the 3l nontranslated region of the bc1-2
transcript (Cleary et al.,~47 Cell 19, 1986). The bc1-2
,,, - ,
gene repre~sents a new form~of proto-oncogene in`that it
encodes a ~mitochondrial protein which inhibits cell
senescence (Hockenbery et al., 348 Nature 334, 1990),
leading to extended survival of B-cells transfected with
this gene (Nunez et al., 86 Proc. Natl. Acad. Sci. USA
4589, 1989).
At least three different forms of bc1-2 mRNAs
are found 1n~pre-B-cells and T-cells, which vary due to
alternative splicing and;promoter usage. Two different
proteins are produced, a 21 kD and a 26 kD peptide which
vary at their carboxytermini. Both forms have identical
... .
93/23057 ,~ 3~ PCT/US93/04573 ;~
13
N termini encoded in exon 2 of the gene. Consequently,
this region would be suitable for ribozyme targeting. -
Breast Cancer ~;
The epidermal growth factor (EGF) receptors have
been implicated in human cancer more frequently than any
other family of growth factor receptors. The EGF receptor
gene is often amplified or overexpressed in squamous cell
carcinomas and glioblastomas. Jenkins et al., 39 Cancer `
Genet. C~toaenet. 253, 1989. Similarly, erbB-2 ls often `~
10 overexpressed in adenocarcinomas of the stomach, breast ~-~
and ovary. Turc-Carel et al., 12 ibid. 1, 1984.
Overexpression of either gene under appropriate
experimental conditions confers the transformed phenotype.
Heim et al., 32 ibid. 13, 1988. In certain breast
carcinomas, the erbB-3 gene is overexpressed. Boehm et
al., 7 EMBO J. 385, 1988.
The high incidence of human breast cancer has
prompted efforts to model the disease in transgenic mice.
The myc gene is amplified in some human breast cancers,
2~0~Escot ;et ~al.~, 83 Proc. Natl. Acad. Sci. USA 4834, 1986,
and ras mutations have been observed. Barbacid, 56 Ann.
Rev. Biochem. 779, 1987.~ Reproduct~ion of disease by
~expresslon of the myc or ras genes in mice have given only
-~ ~ sporadlc~ results.
~ ~ Breast cancer progression often correlates with
amplification of the tyrosine kinase receptor gene denoted
as c-erb-B2 or neu. The ligand for this receptor is
unknown. Male and female mice expressing the neu gene
both synchronously developed adenocarcinomas encompassing
the entire gland.` Muller èt; al., 54 Cell 105, 1988.
Other strains developed tumors stochastically. Bouchard
et al., S7 ibid. 931, 1989.
-Cs~sEL5~_ cinoma
,~ .
- The~platelet derived growth factor (PDGF) system
has served as a prototype for identification of substrates
of the receptor tyrosine kinases. Certain enzymes become
;~ activated by the PDGF receptor kinase, including
':
,
W093/23057 PCT/US93/045
14
phospholipase C and phosphatidylinositol 3' kinase, Ras
guanosine triphosphate (GTPase) activating protein (GAP)
and src-like tyrosine kinases. GAP regulates the function
of the Ras protein. It stimulates the GTPase activity of
the 21 kD Ras protein. Barbacid, 56 Ann. Rev. Biochem.
779, 1987. Microinjection of oncogenically activated Ras
into NIH 3T3 cells induces DNA synthesis. Mutations that
cause oncogenic activation of ras lead to accumulation of
Ras bound to GTP, the active form of the molecule. These
mutations block the ability of GAP to convert Ras to the
inactive form. Mutations that impair the interactions of
Ras with GAP also block the biological function of Ras.
While a number of ras alleles exist (N-ras, K-
ras, H-ras) which have been implicated in carcinogenesis,
the type most often associated with colon and pancreatic
carci:nomas is~the K-ras. Ribozymes which are targeted to
certain regions of thé K-ras allelic mRNAs may also prove
inhibitory t;o the functlon of the other allelic mRNAs of
; the N-ras~and~H-ras genes.
; 20-~Lun~ Cancer/L-mYc Gene
Expression of the myc oncogene is known to alter
cell growth in a number of tissues. The product of this
gene lS~ ~a protein which is known to be a transcriptional
activator that can act singly or in combination with other
oncogene~ proteins. The L-myc gene is often activated by
tra~slo~ations of DNA from other regions of the genome to
the~regulatory regions 5' of the m~c gene ORF. After
transcription of the L-myc mRNA, alternate splicing of the
., .
transcript is known to occur. Kaye et al., 8 Mol. Cell
30 ~Biol. 196, 1988. !` The ai~ternate mRNAs produced contai~ 'a
common 5' exon 1 and portions of a common exon 2. These
common regions of m~NA structure allow the use of nucleic
acid targeted therapeutics which can inactivate both
species of mRNA with one therapeutic molecule.
,,: , .
PromYeloc~tic Leukemia
Acute promyelocytic leukemia is characterized by
~- a specific translocation, a t(15;17)(q22;qll.2-12), which
~ :' `
` ~ o g3/23~57 ~ ~ 3 ~J~,~g PCT/US93/0457~
~5
is found in some 90% of the cases. The t(lS;17) is often
the only detectable cytogenetic abnormality present in the
leukemic cells. This rearrangement results in the fusion
of two genes, the promyelocytic leukemia gene t PML) on
chromosome 15, and the retinoic acid receptor alpha gene
(RARA) on chromosome 17 (J. Borrow et al., 249 Science
1577, 1990; H. de Thé et al., 347 Nature 558, 1990). The
RARA is a hormonally-responsive transcriptional regulatory
protein, while the function of the PML is as yet unknown.
A fusion message is expressed in the leukemic
cells which encodes the N-terminal coding region of the
PML gene and the C-terminal coding region of the RARA
gene. Expression of this fusion gene apparently inhibits
normal myeloid differentiation. The biological relevance
lS of this rearrangement to the etiology of the disease has
been exemplified by the discovery that all-trans retinoic
acid can~be used to achieve complete clinical remission,
presumàbly~by~inducing differentiation of the leukemic
cells. ;This suggests that the fusion protein is still
hormonally responsive.
, .
The treatment of leukemic cells with retinoic
-~ acid is not preferable over the long term because retinoic
acld is a generalized inducer of differentiation in all
cell ~types, ~not just leukemic cells. Thus, systemic
;25~adminl~stration of these compounds can lead to a number of
deleterious side effects by differentiating cells which
should not be~ in a differentiated state. A treatment
which gi~es suppression of the transformed phenotype in
leukemic cells without affecting other cell types is
preferable, as deslcribed herein.
B-Cell Acute LYm~hocv_ic Leukemia
Leukemia comprises some 3% of the new cancer
;~ - cases~per year, with lymphocytic leukemias accounting for
approximately half (National Cancer Institute, 1990
~; 35 statistics). A subset of lymphocytic leukemias of the
;acute pre-B-cell type are associated with a specific
chromosomal translocation, a t(l;l9)(q23;pl3.3) (M.B.
WO93/~30s7 ~l~ 5 4 9 3 PCT/US93/045
16
~amps et al., 60 Cell 547, 1990; J. Nourse et al., ibid.
p.535). This rearrangement results in the fusion of two
genes, the PBX gene present on chromosome 1, and the E2A
gene present on chromosome 19. While the E2A transcript
is found in all B-cell types, the PRL gene is not normally
expressed in B-cells. However, an E2A/PRL fusion message
is constitutively expressed from this aberrant locus in
the leukemic cells. This fusion message encodes the N-
terminal region of the E2A, including the transcriptional
activating domain of that gene, and the C-terminal region
of the PRL gene, which contains a homeodomain DNA binding
motif. Thus, a potentially functional chimeric
transcriptional regulatory protein is expressed in the
leukemic cells.
15The PRL sequences found in the fusion mRNA are
: ~ good targets for ribozyme therapy since PRL iS not
~- ~expressed~ in non-leukemic B-cells. Whether the E2A
sequènc~ès~ can ~be targeted by ribozymes is unclear since
such~ribozymes~may inhlbit E2A expression in normal B-
20~ ~cells.~It~is~not known how normal B-cells are affected by
inhibition of E2A.
me following is but one example of a method by
which suitable target sltes~can be identified and is not
~ miting ~ln~this invention. Generally, the method
s~ 25~ ~involve~s~ identlfylng potential cleavage sites for a
hammerheàd~ribozyme, and then testing each of these sites
to~determine their suitability as targets by ensuring that
secondary structure formation is minimal.
The mRNA sequences are compared in an
appropriate target region. Putative ribozyme cleavage
sites are identified from weak or non-base paired regions
of the mRNA. These sites represent the preferred sites
for hammerhead or other ribozyme cleavage within these
target mRNAs.
,. . ~
~ ~ Short RNA substrates corresponding to each of
the mRNA sites are designed. Each substrate is composed
- of two to three nucleotides at the 5~ and 3~ ends that
;. ~93/23057 ~ PCT/US93/0~573 ~s`
will not base pair with a corresponding ribozyme
recognition region. The unpaired regions flank a central
region of 12-14 nucleotides to which complementary arms in
the ribozyme are designed.
The structure of each substrate sequence is
predicted using a PC fold computer program. Sequences
which give a positive free energy of binding are accepted.
Sequences which give a negative free ener~y are modified
by trimming one or two bases from each of the ends. If
the modified sequences are still predicted to have a
strong secondary structure, they are rejected.
After substrates are chosen, ribozymes are
designed to each of the RNA substrates. Ribozyme folding
is also analyzed using PC fold.
Ribozyme molecules are sought which form
,~ hammerhead motif stem II (see Fig. 1) regions and contain
flanking~arms which are devoid of intramolecular base
pairing. Often t-he ribozymes are modified by trimming a
base f rom the~ ends of the rlbozyme, or by introducing
~add~ltional~base pairs in stem II to achieve the desired
f~old~ Ribozymes with incorrect folding are rejected.
After substrate/ribozyme pairs are found to contain
correct~ intramolecular structures, the molecules are
foldedl together to predict 1ntermolecular interactions.
-~ ~ 25 A schematic representation of a ribozyme with its
coordlnate base pairing to its cognate target sequence is
shown in~Fig~ 1. Examples of useful targets are listed in
Figs. 2
Those targets thought to be useful as ribozyme
targets can be tested to determine accessibility to
~- nucleic acid probes in a ribonuclease H assay (see below).
This assay provides a quick test of the use of the target
site without requiring synthesis of a ribozyme. It can be
used to~screen~for~sites most suited for ribozyme attack.
35~ SYnthesis of Ribozymes
Ribozymes useful in this invention can be
; produced by gene transcription as described by Cech,
.,
'''
WO93/230s7 ~ g ~CT/US93/045
18
supra, or by chemical synthesis. Chemical synthesis of
RNA is similar to that for DNA synthesis. The additional
2~-OH group in RNA, however, requires a different
protecting group strategy to deal with selective 3'-5'
internucleotide bond formation, and with RNA
susceptibility to degradation in the presence of bases.
The recently developed method of Z~NA synthesis utilizing
the t-butyldimethylsilyl group for the protection of the
2' hydroxyl is the most reliable method for synthesis of
ribozymes. The method reproducibly yields RNA with the
correct 3'-5' internucleotide linkages, with average
coupling yields in excess of 99%, and requires only a two-
step deprotection of the polymer.
A method based upon H-phosphonate chemistry of
phosphoramidites gives a relatively lower coupling
efficiency than a method based upon phosphoroamidite
chemis~try.~ Thls is a problem ~for synthesis of DNA as
well~ A~ promising approach to scale-up of automatic
ol~igonuc-leotide~synt~e~sls has been described recently for
2~0 the~ H~-phosphonates~. A combination of a proper coupling
time~and~additional capping of ~Ifailure~ sequences gave
high yields in the synthesis of oligodeoxynucleotides in
scales ~in~ the range of 14 -~moles with as little as 2
e~uivalents of a~monomer in the coupling step. Another
2S~alternatlve~approach lS to use soluble polymeric supports
polyethylene glycolsj, instead of the conventional
;solid ~ ~supports. This method can yield short
oligonucleotides in hundred milligram quantities per batch
utiliz~ng about 3 equivalents of a monomer in a coupl1ng
30~ step. `
Various modifications to ribozyme structure can
be made to enhance the utility of ribozymes. Such
--~ modifications~will enhance shelf-life, half-life in vitro,
stability, and ease of introduction of such ribozymes to
35 ~the targ~et site, s~g~, to enhance penetration of cellular
membranes, and confer the ability to recognize and bind to
targeted cells.
:~ , ;
~93/23057 PCT/US93/0457~ ~
1 9 . :
Exogenous delivery of ribozymes benefits from
chemical modification of the backbone, e.q., by the
overall negative charge of the ribozyme molecule being
reduced to facilitate diffusion across the cell membrane.
The present strategies for reducing the oligonucleotide
charge include: modification of internucleotide linkages
by methylphosphonates, use of phosphoramidites, linking
oligonucleotides to positively charged molecules, and
creating complex packages composed of oligonucleotides,
lipids and specific receptors or effectors for targeted
cells. Examples of such modifications include sulfur-
containing ribozymes containing phosphorothioates and
phosphorodithioates as internucleotide linkages in RNA.
Synthesis of such sulfur-modified ribozymes is achieved by
use of the sulfur-transfer reagent, 3H-1,2-benzenedithiol-
3-one 1,1-dioxide. Ribozymes may also contain ribose
modified ribonucleotides. Pyrimidine analogues are
prepared from uridine using a procedure employing
- diethylamino sulphur trifluoride ~DAST) as a starting
20 ~reagent~. ~ Ribaz ~es can also be either electrostatically
-~ or covalently attached to polymeric cations for the
purpose of reducing charge. The polymer can ~e attached ~-
- !~'
~ to the ribozyme by simply converting the 3'-end to a
- ~ ribonucleoside dialdehyde which is obtained by a periodate
cleavage of the terminal 2~,3~-cis diol system. Depending
on the specific requirements for delivery systems, other
possible modifications may include different linker arms
containing carboxyl, amino or thiol functionalities. Yet
further~examples include use of methylphosphonates and 2'-
O-methylribose and 5' or 3' capping or blocking with
m7GpppG or m32~7GpppG.
For example, a kinased ribozyme is contacted
with guanosine triphosphate and guanyltransferase to add
a m3G cap to the ribozyme~ After such synthesis, the
ribozyme can be gel purified using standard procedure. To
ensure that the ribozyme has the desired activity, it may
be tested with and without the 5' cap using standard
..
WC) 93/ 230 !7 PCr/ US93/0457
`3 9
procedures to assay both its enzymatic activity and its
stability.
Synthetic ribozymes, including those containing
various modifiers, can be purified by high pressure liquid
chromatography tHPLC). Other liquid chromatography
techniques, employing reverse phase columns and anion
exchangers on silica and polymeric supports may also be
used.
There follows an example of the synthesis of one
ribozyme. A ~olid phase phosphoramidite chemistry was
employed. Monomers used were 2'-tert-butyl-dimethylsilyl
cyanoethylphosphoramidities of uridine, N-benzoyl-
cytosine, N-phenoxyacetyl adenosine and guanosine (Glen
Research, Sterling, VA). Solid phase synthesis was
carried out on either an ABI 394 or 380B DNA/RNA
synthesizér using the standard protocol provided with each
machine. ~The only~exception~ was that the coupling step
was increased f~rom 10 to 12 minutes. The phosphoramidite
concentratlon was 0.1 M. Synthesis was done on z 1 ~mole
20~ sc~alè~uslng~a~l ~ le RNA reaction column (Glen Research).
The~average~coupling efficiencies were between 97% and 98%
for~the 394 model, and between 97% and 99% for the 380B
model, ~as determined by a calorimetric measurement of the
released trltyl cation.
25~ Blocked ribozymes were cleaved from the solid
support (~ , CPG), and the bases and diphosphoester
moiety ~deprotected in a sterile vial by dry ethanolic
ammonia (2 mL) at 55C for 16 hours. The reaction mixture
was cooled on dry ice. Later, the cold liquid was
transferred into a sterile screw cap vial and lyophi`lized.
To remove the 2'-tert-butyl-dimethylsilyl groups
from the ribozyme, the residue was suspended in 1 M tetra-
n-butylammonium fluoride in dry THF (TBAF), using a 20-
" .
fold ex~ess of the reagent for every silyl group, for 16
~- 35 hours at ambient temperature (about 15-25C). The
reaction was quenched by adding an equal volume of sterile
.
93/23057 ~ 4 ~ ~ PCT/US93/04~7
21
1 M triethylamine acetate, pH ~.5. The sample was cooled
and concentrated on a SpeedVac to half the initial volume.
The ribozymes were purified in two steps by HPLC
on a C4 300 A 5 mm DeltaPak column in an acetonitrile
gradient.
The first step, or ~trityl on" step, was a
separation of 5'-DMT-protected ribozyme(s) from failure
sequences lacking a 5'-DMT group. Solvents used for this
step were: A tO.1 M triethylammonium acetate, pH 6.8~ and -
B (acetonitrile). The elution profile was: 20% B for 10
minutes, followed by a linear gradient of 20% B to 50% B
over 50 minutes, 50% B for 10 minutes, a linear gradient
of 50% B to 100% B over 10 minutes, and a linear gradient
~ ~ .
of 100% B to 0% B over 10 minutes.
15The ~second step was a purification of ~a
completely deblocked ribozyme by a treatment of 2%
trifluoroacetlc acid on a C4 300 A 5 mm DeltaPak column in
an ac~etonitrile gradient. Solvents used for this second ---
step were:~ A ~0.1 M triethylammonium acetate, pH 6.8) and ',!
20 ~B~(80%~ acetonitrile, 0.1 M triethylammonium ~acetate, pH
6.~8~ The elution profile was: 5% B for 5 minutes, a
linear ~gradient of 5% B to 15% B over 60 minutes, 15% B :~"
for 10~mlnutes, and a linear gradient of 15% B to 0% B j-~
over 10 minutes. l~
25~ The fraction containing ribozyme was cooled and r','
lyophillzed on a SpeedVac. Solid residue was dissolved in
a minimum amount; of ethanol and sodium perchlorate in
acetone. The ribozyme was collected by centrifugation, ~
washed three times with acetone, and lyophilized. ~`
ExPression Vector
While synthetic ribozymes are preferred in this -~
invention, those produced by expression vectors can also
be used. In designing a suitable ribozyme expression
vector the following factors are important to consider. ~-
The final ribozyme must be kept as small as possible to
minimize unwanted secondary structure within the ribozyme. -
~- A promoter (e.q., a T7, human cytomegalovirus immediate
,,
W093/23057 PCT/US93/0457'~
22
early (iel), human beta actin, or U6 snRNA promoters)
should be chosen to be a relatively strong promoter, and
expressible both in vitro and in vivo (e.q., by co-
infection with the T7 RNA polymerase gene, the human
cytomegalovirus immediate early (iel) or human beta actin
promoters). Such a promoter should express the ribozyme ~-
at a level suitable to effect production of enough
ribozyme to destroy a target RNA, but not at too high a
level to prevent other cellular activities from occurring
(unless cell death itself is desired).
A hairpin at the 5' end of the ribozyme is
useful to ensure that the required transcription
initiation sequence (GG or GGG or GGGAG) does not bind to
some other part of the ribozyme and thus affect regulation -
of the transcription process. The 5' hairpin is also
useful to protect the ribozyme from 5'-3' exonucleases. ~`~
~ A~selected~halrpln at the 3' end of the ribozyme gene is
-~ useful since it acts as a transcription termination
slgnal, and~ protects the ribozyme from 3'-5' exonuclease
~ 20 actlvity. One example of a known termination signal is
r~ that~present on the T7 RNA polymerase system. This signal
i;s~about~30~ nucleotides in length. Other 3' hairpins of
shorter length can be used to provide good termination and
RNA stability. Such hairpins can be inserted within the ~:
vector sequences to allow standard ribozymes to be placed
in an ~appropriate orientation and expressed with such
sequences attached.
Poly(A) tails are also useful to protect the 3'
end of the ribozyme. These can be provided by either
including 'a poly(A) signal slte in the expre~ssion vector'
(to signal a cell to add the poly(A) tail in vivo), or by
~ -~ introducing a poly(A) sequence directly into the
- - expression vector. In the first approach the signal must
- be located to prevent unwanted secondary structure
formation with other parts of the ribozyme. In the second
appr~ach, the poly(A) stretch may reduce in size over time
when expressed in vivo, and thus the vector may need to be
, .
:: .
9 9
93/230~7 PCT/US93/0457
checked over time. Care must be taken in addition of a
poly(A) tail which binds poly~A) binding proteins which
prevent the ribozyme from acting.
RibozYme Tes inq
Once the desired ribozymes are selected,
synthesized and purified, they are tested in kinetic and
other experiments to determine their utility. An example
of such a procedure is provided below.
Pre~aration of Ribozvme
Crude synthetic ribozyme (typically 350 ~g at a
time) was purified by separation on a 15% denaturing
polyacrylamide gel (0.75 mm thick, 40 cm long) and
visualized by W shadowing. Once excised, gel slices
containing full length ribozyme were soaked in 5 ml gel
elution buffer (0.5 M NH40Ac, 1 mM EDTA) overnight with
shaking at 4C. The eluent was desalted over a C-18
matrix (Sep-Pak cartridges, Millipore, Milford, MA) and
vacuum dried. The dried RNA was resuspended in 50-100 ~1
~ TE (TRIS 10 mM, EDTA 1 mM, pH 7.2). An aliquot of this
--f~ 20 ~so~1ution was diluted 100-fold into 1 ml TE, half of which
was used to spectrophotometrically quantitate the ribozyme
solution. The concentration cf this dilute stock was
typically 150~800 nM. Purity of the ribozyme was
confirmed by the presence of a single band on a denaturing
- .
polyacrylamide gel.
A ribozyme may advantageously be synthesized in
two or more portions. Each portion of a ribozyme will
generally have only limited or no enzymatic activity, and
the actlvity will increase substantially (by at least 5-10
fold) when all portions are ligated (or otherwise
juxtaposed) together. A specific example of hammerhead
ribozyme synthesis is provided below.
~ .
- The method involves synthesis of two (or more)
shorter "half" ribozymes and ligation of them together
using T4 RNA ligase. For example, to make a 34 mer
ribozyme, two 17 mers are synthesized, one is
phosphorylated, and both are gel purified. These purified
w093/~3057 ~,~ 3~4~3 PCT/U593/045
24
17 mers are then annealed to a DNA splint strand
complementary to the two 17 mers. (Such a DNA splint is
not always necessary.) This DNA splint has a sequence
designed to locate the two 17 mer portions with one end of
each adjacent each other. The juxtaposed RNA molecules
are then treated with T4 RNA ligase in the presence of
ATP. The 34 mer RNA so formed is then HPLC purified.
Preparation of Substrates
Approximately 10-30 pmoles of unpurified
substrate was radioactively 5' end-labeled with T4
polynucleotide kinase using 25 pmoles of [~_32p] ATP. The
entire labeling mix was separated on a 20% denaturing
polyacrylamide gel and visualized by autoradiography. The
full length band was excised and soaked overnight at 4C
in 100 ~l of TE (10 mM Tris-HCl pH 7.6, 0.1 mM ~DTA).
Kinetic Reactions
For reactions using short substrates (between 8
and 16 bases) a substrate solution was made lX in assay
; buf~er (75 mM Tris-HCl, pH 7.6; 0.1 mM EDTA, 10 mM MgCl2)
such that~ the concentration of substrate was less than
1 nM. A ribozyme solution (typically 20 nM) was made lX
in assay buffer and four dilutions were made using lX
assay buffer. Fifteen ~l of each ribozyme dilution (i.e.,
20, 16, 12, 8 and 4 nM) was placed in a separate tube.
These tubes and the substrate tube were pre-incubated at
37C for at least five minutes.
The reaction was started by mixing lS ~l of
substrate into each ribozyme tube by rapid pipetting (note
that final ribozyme concentrations were 10, 8, 6, 4,
2 nM). Five ~l aliquots were removed at 15 or 30 second
intervals and quenched with 5 ~l stop solution (95%
formamide, 20 mM EDTA xylene cyanol, and bromphenol blue
dyes~. Following the final ribozyme time point, an
aliquot of the remaining substrate was removed as a zero
ribozyme control.
The samples were separated on either 15% or 20%
polyacrylamide gels. Each gel was visualized and
~093~3057 PCT/US93/0457
quantitated with an Ambis beta scanner (Ambis Systems, San
Diego, CA).
For the most active ribo~ymes, kinetic analyses
were performed in substrate excess to determine ~ and Kcat
values.
For kinetic reactions with long RNA substrates
(greater than 15 bases in length) the substrates were
preparéd by transcription using T7 RNA polymerase and
defined templates containing a T7 promoter, and DNA
encoding appropriate nucleotides of the target RNA. ~he
substrate solution was made lX in assay buffer ~7S mM
Tris-HCl, pH 7.6; 0.1 mM EDTA; 10 mM MgCl2) and contained
58 nanomolar concentration of the long RNA molecules. The
reaction was started by addition of gel purified ribozymes
15 to 1 ~M concentration. Aliquots were removed at 20, 40,
; ~ 60, 80 and 100 minutes, then quenched by the addition of
5 ~1 stop solution. Cleavage products were separated
using~denaturing PAGE. The bands were visualized and
quant~1tat~ed with~an Ambis beta scanner.
Kinetic Analysis
;~ A simple reaction mechanism for ribozyme-
mediated cleavage is:
ki k~
- ~ ~
~ R~ S ~ [R:S] ~ [R:P] I ~ R + P
- k l
where R = ribozyme, S = substrate, and P = products. The
boxed step is important only in substrate excess. Beca~se
ribozyme concentration, is~ in excess over substrate
concentration, the concentration of the ribozyme-substrate
complex (lR:S]) is constant ov-~r time except during the
very brief time when the complex is being initially
,.
~; formed, i.e.,:
-~ 35 d~R:Sl = 0
-~ dt
where t = time, and thus:
~- (R)(S)~' = (RS~(k~ + k,).
~,'', ' ,
c~
W(~ 93/230 ~7 ~ I v PCr/US93/0457 r 5
26 `
The rate of the reaction is the rate of disappearance of
substrate with time:
Rate = (t) = k2(RS)
., .
Substituting these expressions:
(X)(S)k~ = 1/k2 -d(S) ~k2 + k,)
dt
or:
-d(S) = _ k,k._ (R) dt
S (k2 ~ k,)
Integrating this expression wlth respect to time yields:
-ln S = k1k~ ~R) t
So ( k2 ~ kl )
where S0 = inltial substrate. Therefore, a plot of the ;~
negative log of fraction substrate uncut versus time (in
minutes) yields a straight line with slope:
,
slope = k1k. (R) = kObs
~k2 + k,)
where kob5 = observed rate constant. A plot of slope (kob5)
20 versus ribozyme concentration yields a straight line with
a slope which is:
slope = k.k which is k
(k7 ~ k1) ~
Using these equations the data obtained from the
,
25 kinetic experiments provides the necessary information to
determine which ribozyme tested is most useful, or active.
Such ribozymes can be selected and tested in in vivo or
èx vivo systéms. ~ ! `! ' `;. ! ' :
Liposome Preparation
Lipid molecules are dissolved in a volatile
organic solvent (CHCl3, methanol, diethylether, ethanol,
etc.). The organic solvent is removed by evaporation.
The lipid is hydrated into suspension with O.lx phosphate
buffered saline (PBS), then freeze-thawed 3x using liquid
nitrogen and incubation at room temperature. The
~;~
..~9~t23057 ~ 1~ 3 4 9 9 PC~tUS93tO4~7~
,;.
27
suspension is extruded sequentially through a 0.4 ~m,
O.2 ~m and 0.1 ~m polycarbonate filters at maximum
pressure of 800 psi. The ribozyme is mixed with the .-
extruded liposome suspension and lyophilized to dry~^ess. ~-
The lipid/ribozyme powder is rehydrated with water to one-
tenth the original volume. The suspension is diluted to
the minimum volume required for extrusion (0.4 ml for
1.5 ml barrel and 1.5 ml for 10 ml barrel) with lxPBS and
re-extruded through 0.4 ~m, 0.2 ~m, 0.1 ~m polycarbonate
filters. The liposome entrapped ribozyme is separated
from untrapped ribozyme by gel filtration chromatography
(SEPHAROSE CL-4B, BIOGEL A5M). The liposome extractions
are pooled and sterilized by filtration through a 0.2 ~m
filter. The free ribozyme is pooled and recovered by
ethanol prec}pitation. The liposome concentration is
determined by incorporation of a radioactive lipid. The
ribozyme concentration is determined by labeling with 32p,
Rossi et al., 1992, supra (and references cited therein)
; describe other methods suitable for preparation of
liposomes.
Examples of other useful liposome preparations
which display similar degrees of upt~ke of both a
radioactive lipid marker and an entrapped fluorophore by
Vero cells showed different fluorescent staining patterns.
- 25 Specifically, liposomes composed of DPPG/DPPC/Cholesterol
~- (in a ratio of: 50/17/33) gave a punctate pattern of
fluorescence, while DOPE/Egg PC/Cholesterol (30/37/33)
gave a diffuse, homogeneous pattern of fluorescence in the
cytoplasm. Cell fractionation showed that 80% of the
entrapped contents from the DPPG/DPPC/Cholesterol
formulation was localized in the membrane fraction,
whereas the DOPE/Egg PC/Cholesterol formulation was
localized in the cytoplasm. Further characterization of
the latter formulation showed that after 3 hours, 70% of
the fluorescence was cytoplasmic and 30% was in the
membrane. After 24 hours, uptake had increased 5-fold and
W~l93/23057 ~ 3~4 PCI/IS93/0457'~
28
the liposome contents were distributed 50/50 between the
cytoplasmic and membrane fractions.
Liposomes containing 15 ribozymes (32P-labeled)
targeted to the HSV ICP4 mRNA were prepared and incubated
with the cells. After 24 hours, 25% of the liposome dose
was taken up with approximately 60,000 liposomes per cell.
Thirty percent of the delivered ribozyme was intact after
24 hours. Cell fractionation studies showed 40% of the
intact ribozyme to be in the membrane fraction and 52% of
the intact ribozyme to be in the cytopIasmic fraction.
In Vi vo Assav
The efficacy of action of a chosen ribozyme may
be tested in vivo by use of cell cultures sensitive to
mdr-1 gene expression, using standard procedures in
transformed cells or animals which express the target mRNA
- using standard procedures.
The efficacy of action of a chosen ribozyme may
be tested in t~ssue culture by use of transformed cells
containing the target mRNA (e.q., K562 cells which express
~;~ 20 the b3;a2~ ~fusion mRNA) using standard procedures.
Alternatively, ribozyme efficacy could be tested with
peripheral blood or bone marrow from CML patients using
soft-agar colony forming assays. Such methods are known
-
to those educated in this field.
Ribonuclease Protection AssaY
~ The accumulatlon of target-mRNA in cells or the
- cleavage of the mRNA by ribozymes or RNaseH (in vitro or
in vivo) can be quantified using an RNase protection
assay.
In this method, antisense riboprobes are
transcribed from template DNA using T7 RNA polymerase
(U.S. Biochemical) in 20 ~l reactions containing lX
transcription buffer (supplied by the manufacturer),
0.2 mM ATP, GTP and UTP, 1 U/~l pancreatic RNase inhibitor
35 (Boehringer Mannheim 8iochemicals) and 200 ~Ci 3~P-labeled
CTP (800 Ci/mmol, New England Nuclear) for 1 hour at 37C.
Template DNA is digested with 1 U RNase-free DNaseI (U.S.
.. 093/23057 ~1 3 ~ PCT/US93/0457~ ~
'~:
29 ~`
Biochemical, Cleveland, OH) at 37C for 15 minutes and
unincorporated nucleotides removed by G-50 SEPHADEX spin
chromatography.
In a manner similar to the transcription of
antisense probe, the target mRNA can be transcribed
in vi tro using a suitable DNA template. The transcript is
purified by standard methods and digested with ribozyme at
37C according to methods described later.
Alternatively, afflicted ~mRNA-expressing~ cells
expressing the target mRNA bcr-abl fusion transcript are
harvested into 1 ml of PBS, transferred to a 1.5 ml
EPPENDORF tube, pelleted for 30 seconds at low speed in a
microcentrifuge, and lysed in 70 ~l of hybridization
buffer (4 M guanidine isothiocyanate, 0.1% sarcosyl, 25 mM
sodium citrate, pH 7.5). Cell lysate (45 ~l) or defined
amounts of in vitro transcript (also in hybridization
buffer) is then combined with 5 ~l of hybridization buffer
containing 5 x 105 cpm of each antisense riboprobe in 0.5
ml EPPENDORF tubes, overlaid with 25 ~l mineral oil, and
hybridization accomplished by heating overnight at 55C.
The hybridization reactions are diluted into 0.5 ml RNase
solution (20 U/ml RNaseA, 2 U/ml RNaseTl, 10 ~/ml RNase-
free DNaseI in 0.4 M NaCl), heated for 30 minutes at 37C,
and 10 ~l of 20% SDS and 10 ~l of Proteinase K (10 mg/ml)
added, followed by an additional 30 minutes incubation at
: 37C. Hybrids are partially purified by extraction with
0.5 ml of a 1:1 mixture of phenol/chloroform; aqueous ;.
phases are combined with 0.5 ml isopropanol, and RNase- :
resistant hybrids pelleted for 10 minutes at room
temperature (about 20C) in a microcentrifuge. Pellets
are dissolved in 10 ~l loading buffer (95% formamide, lX :
TBE, 0.1% bromophenol blue, 0.1% xylene cylanol), heated
to 95C for five minutes, cooled on ice, and analyzed on
4~ polyacrylamide/7 M urea gels under denaturing :
conditions.
WOg3/230~7 ~13 ~ ~ 9 ~ PCTtUS93/045
Ribozvme Stabilitv
The chosen ribozyme can be tested to determine
its stability, and thus its potential utility. Sush a
test can also be used to determine the effect of various
chemical modifications (e.q., addition of a poly(A) tail)
on the ribozyme stability and thus aid selection of a more
stable ribozyme. For example, a reaction mixture contains
1 to 5 pmoles of 5~ (kinased) and/or 3' labeled ribozyme,
15 ~g of cytosolic extract and 2.5 mM MgCl2 in a total
volume of 100 ~1. The reaction is incubated at 37C.
Eight ~1 aliquots are taken at timed intervals and mixed
- wlth 8 ~l of a stop mix (20 mM EDTA, 95% formamide).
Samples are separated on a 15% acrylamide sequencing gel,
exposed to film, and scanned with an Ambis.
A 3'-labeled ribozyme can be formed by
-~ incorporation of the 32P-labeled cordycepin at the 3~ OH
.
- using poly(A) polymerase. For example, the poly(A)
polymerase reaction contains 40 mM Tris, pH 8, 10 mM MgCl2,
25~0 mM NaCl, 2.5 mM MnCl2; 3 ~l 32p cordycepin, 500 Ci/mM;
20 ~and 6 unlts poly(A) polymerase in a total volume of 50 ~1.
The reaction mlxture is incubated for 30 minutes at 37C.
Effect of Base Substitution u~on Ribozvme
~ctivity
To determine which primary structural
characteristics could change ribozyme cleavage of
substrate, minor base changes can be made in the substrate
cleavage region recognized by a specific ribozyme. For
example, the substrate sequences can be changed at the
central~nC" nucleotide, changing the cleavage site from a
GUC to a GUA motif. The KCat/~ values for cleavage using
each substrate are then analyzed to determine if such a
`~- change increases ribozyme cleavage rates. Similar
M ~ experiments can be performed to address the effects of
changing bases complementary to the ribozyme binding arms.
Changes ~predicted to maintain strong binding to the
complementary substrate are preferred. Minor changes in
nucleotide content can alter ribozyme/substrate
~093/230~7 ` ~ 5 (~ !i 3 PCT/VS93/04573 ~i
(.
31
interactions in ways which are unpredictable based upon
binding strength alone. Structures in the catalytic core
region of the ribozyme recognize trivial changes in either
substrate structure or the three dimensional structure of
the ribozyme/substrate complex.
To begin optimizing ribozyme design, the
cleavage rates of ribozymes containing varied arm lengths,
but targeted to the same length of short RNA substrate can
be tested. Minimal arm lengths are required and effective
cleavage varies with ribozyme/substrate combinations.
The cleavage activity of selected ribozymes can
be assessed using target mRNA-homologous substrates. The
assays are performed in ribozyme excess and approximate
Kcae/Kmin values obtained. Comparison of values obtained
with short and long substrates indicates utility in ~-ivo
of a ribozyme.
Intracellular Stabilitv of LiPosome-Delivered
ibozvmes
,
To test the stability of a chosen ribozyme
- 20 :in vivo the following test is useful. Ribozymes are 32p_
end-labeled, entrapped in liposomes and delivered to
; target mRNA containing cells for three hours. The cells
are ~fractionated and ribozyme is purified by
phe~ol~chloroform extraction. Alternatively, cells ( lX107,
T-I75 flask) are scraped from the surface of the flask are
~- cultured and washed twice wlth cold PBS. The cells are
. .
homogenized by douncing 35 times in 4 ml of TSE (10 mM
Tris, pH 7.4, O.25 M Sucrose, mM EDTA) . Nuclei are
pelleted at lOOxglfo~ 10 minutes. Subcellular organelles
(the m~ ~ane fraction) are pelleted~at 200,000xg for two
- hours ; ng an SW60 rotor. The pellet is resuspended in
1 ml of H buffer (0.25 M Sucrose, 50 mM HEPES, pH 7.4).
The supernatant contains the cytoplasmic fraction (in
; approximately 3 . 7 ml ) . The nuclear pellet is resuspended
in 1 ml of 65% sucrose in TM (50 mM Tris, pH 7.4, 2.5 mM
MgCl2) and banded on a sucrose step gradient (1 ml nuclei
~`~ in 65% sucrose TM, 1 ml 60% sucrose TM, 1 ml 55% sucrose
W09~/230~7 ' ~ 3 ~ 4 3 ~ PCT/US93/~4~7s. ` ~
.
32
TM, 50% sucrose TM, 300 ~l 25% sucrose TM) for one hour at
37,000xg with an SW60 rotor. The nuclear band is
harvested and diluted to 10% sucrose with TM bu~fer.
Nuclei are pelleted at 37,000xg using an SW60 rotor for lS
minutes and the pellet resuspended in 1 ml of TM buffer.
Aliquots are size fractionated on denaturing
polyacrylamide gels and the intracellular localization
determined. By comparison to the migration rate of newly
syntnesized ribozyme, the various fractions containing
10 intact ribozyme can be determined. '
To investigate modifications which would
lengthen the half-life of ribozyme molecules
intracellularly, the cells may be fractioned as above and
the purity~ of each fraction assessed by assaying enzyme '
lS activity known to exist in that fraction. ' ~;
The various cell fractions are frozen at -70C
and used to determine relative nuclease resistances of
modified ribozyme molecules. Ribozyme molecules may be
synthesized wlth 5 phosphorothioate (ps), or 2~-O-methyl
(2~-OMe~modifications at each end of the molecule. These
' molecules and a phosphodiester version of the ribozyme are
end-labeled with 32p and ATP using T4 polynucleotide
kinase. Equal concentrations are added to the cell i-
cytoplasmlc extracts and aliquots of each taken at 10
25 minute intervals. The samples are size fractionated by `~
denaturing PAGE and relative rates of nuclease resistance -
analyzed;by scanning the gel with an Ambis ~-scanner. The
results show whether the ribozymes are digested by the
'cytoplasmic extrac,t,land which versions are relativelyi
more nuclease resistant. Modified ribozymes generally
maintain 80-90% of the catalytic activity of the native
ribozyme when short RNA substrates are employed.
Unlabeled, 5' end-labeled or 3' end-labeled
ribozymes can be used in the assays. These experiments
can also be performed with human cell extracts to verify
the observations.
`'~:
.~093/23057 ~ 1 7~ r~ ~ PCT/US93/0457
In one example, Vero or HeLa cells were grown to
90-95% confluency in 175 cm2 tissue culture flasks, scraped
into 10 ml of cold phosphate buffered saline tPBS), then
washed once in 10 ml of cold PBS and once in 10 ml of cold
TSE (10 mM Tris, pH 7.4; 0.25 M sucrose; 1 mM EDTA). The
cell pellets were resuspended in 4 ml of TSE, dounced 35x
on ice, and the released nuclei pelleted by centrifugation
at lOOOg for 10 minutes. The nuclear pellet was
resuspended in 1 ml of 65% sucrose TM (50 mM Tris, pH 7.4;
2.5 mM MgCl2) and transferred to seckman ultra-clear tubes.
The following sucrose TM solutions were layered on top of
the sample: 1 ml 60%, 1 ml 55~, and 25% sucrose to the
top of the tube. Gradients were spun in an SW60 rotor at
37,0nOg for 1 hour. HeLa nuclei banded at the 55-60%
sucrose boundary and Vero nuclei banded at the 50-55%
sucrose boundary. Nuclear bands were harvested, diluted
to 10% sucrose with TM buffer, and pelleted by
centrifugation at 37,000g for 15 minutes using an SW60
-~ rotor. The nuclear pellet was resuspended in 1 ml of TM
buffer. Subcellular organelles and membrane components in
the post nuclear supernatant were separated from the
cytoplasmic fraction by centrifugation at 200,000g for 2
hours in an SW60 rotor. The pellet contained the membrane
fraction,~ which was resuspended in 1 ml of H buffer (0.25
M sucrose; 50 mM HEPES, pH 7.4), and the supernatant
contained the cytoplasmic fraction.
Purity of the various fractions was assessed
using enæymatic markers specific for the cytoplasmic and
- ~membranous fractions. Three enzyme markers for the
membranous fraction were used; hexosaminidase and ~-
glucocerebrosidase are localized in lysosomes, while
alkaline phosphodiesterase is specific to endosomes.
~- Specifically, the assays were as follows:
~ ,,
-; For N-acetyl-beta-hexosaminidase, the reaction
mixture contained 0.3 mg/ml 4-methylumbelliferyl-N-acetyl-
glucosaminide; 20 mM sodium citrate; pH 4.5; 0.01% Triton
-- X-100; and 100 ~l of sample in a final volume of 500 ~l
W093/~30~7 ~ 4 ~ ~ PCT/US93/04
34
(Harding et al., 6~ Cell 393, 1991). The reactions were
incubated at 37~C for 1 hour and stopped by the addition
of 1.5 ml of stop buffer (0.13 M glycine, 0.07 M NaCl,
0.08 M sodium carbonate, pH 10.6). The reaction product
was quantitated in a Hitachi F-4010 fluorescence
spectrophotometer by excitation of the fluorophore at
360 nm and analysis of the emission at 448 nm.
For Alkaline Phosphodiesterase, the assay medium
contained 25 mM CAPS (3-(Cyclohexylamino)-propanesulfonic
10 acid), pH 10.6; 0.05% Triton X-100; 15 mM MgCl2; 1.25 mg/ml
Thymidine-5~-monophosphate-p~nitrophenyl ester; and 100 ~l
o, sample in a total reaction volume of 200 ~l. The
reactions were incubated at 37C for 2 hours, then diluted
to 1 ml with H2O and the absorbance was measured at 400 nm
15 (Razell and Khorana, 234 J. siol. Chem. 739, 1959).
For ~-glucocerebrosidase, the reaction contained
85 nM sodium citrate, pH 5.9; 0.12% Txiton X-100; 0.1%
sodium taurocholate; 5 mM 4-methylumbelliferyl ~-D-
glucopyranoside; and 125 ~l of sample in a total volume of
- - 20 250 ~l (Kennedy and Cooper, 252 Biochem. J. 739, 198&).
The reaction was incubated at 37C for 1 hour and stopped
by the addition of 0.75 ml of stop buffer. Product
formation was measured in a fluorescence spectrophotometer
~- ~y using an excitation wavelength of 360 nm and analysis
of the emission at 448 nm.
The cytoplasmic enzyme marker, lactate
dehydrogenase, was assayed in an assay mixture containing
O.2 M Tris; pH 7.4; 0.22 mM NADH; 1 mM sodium pyruvate;
and 50 ~l of sample in a final volume of 1.05 ml. Enzyme
"
levels were determined by decreased absorbency at 350 nm
resulting from the oxidation of NADH at room temperature
~- (Silverstein and Boyer, 239 J. Biol. Chem. 3901, 1964).
Lactate dehydrogenase was found predominantly in
the cytoplasmic fractions of both Vero and HeLa cells,
while ~-glucocerebrosidase and alkaline phosphodiesterase
were found almost exclusively in the membranous fractions.
The hexosaminidase activity in Vero cell fractions was
~93/230~7 ~; 3'i~ PCT/US93J0457
concentrated in the membranous fraction (70%) with about
20% in the cytoplasmic fraction. The isolation of enzyme
markers with the appropriate cellular compartment
demonstrated that cytoplasmic, membranous and nuciear
fractions can be isolated with minimal intercompartmental
contamination using this fractionation scheme.
Nuclease StabilitY of Ribozymes and mRNA
The simplest and most sensitive way to monitor
nuclease activity in cell fractions is to use end-labeled
oligonucleotides. However, high levels of phosphatase
activity in some biological extracts gives ambiguous
results in nuclease experiments when 3-P-5~-end-labeled
oligonucleotides are used as substrates. To determine the
phosphatase activity in the extracts, cellular fractions
were incubated with cold ribozymes and trace amounts of
5'-end-labeled ribozyme in the presence of 1 mM Mgt2 (or
- Znt2 with HeLa cytoplasmic extracts) to optimize digestion.
After polyacrylamide gel electrophoresis of samples,
~- digestion~of the oligonucleotide was assessed both by
staining an~d by autoradiography.
Specifically, the basic oligonucleotide
digestion reaction contained substrate nucleic acid (an
RNA ollgonucleotide of 36 nucleotides) and cell fraction
~s~ extract in a total volume of 100 ~ liquots ~7 ~l) were
taken after various periods of incubation at 37C and
added ~to 7 ~l of gel loading buffer (95~ formamide, 0.1
bromophenol blue, 0.1% xylene cyanol, and 20 mM EDTA).
The samples were separated by electrophoresis on a 7 M
urea,~ 20%lj polyaçrylamid~e ,gçl. Intact ribozymes were
visualized either by staining with Stains-all (United
States Biochemical, Cleveland, OH), or autoradiography of
32P-labeled ribozyme. The stained gels and X-ray films
were scanned on a Bio 5000 density scanner (U.S.
Biochemical). Ribozymes were 5' end-labeled with T4
polynucleotide kinase (U.S. Biochemical) using 10 ~Ci of
~- 32p ~-ATP (3,000 Ci/mmole, New England Nuclear, Boston,
MA), and 20-25 pmoles of ribozymes. The unincorporated
'
W093/230~7 ~ 3~ ~ 9 ~ PCT/US93/0457
.
36
nucleotides were separated from the product by G-50 spin
chromatography. Nuclease assays contained 1-2 pmoles of
32p-labeled ribozyme. All oligonucleotides were
synthesized on an Applied Biosystems 394 DNA/RNA
synthesizer (Applied Biosystems Inc., Foster City, CA)
according to manufacturer~ 5 protocols. The nuclear
fractions were resuspended in a buffer containing 2.5 mM
MgCl2. Experiments involving the nuclear fractions were
performed in the presence of 1 mM M~, or in combination
with 1 mM Mn'2, Ca~2, or Zn+2.
To measure the stability of mRNA, Vero cells
were infected with herpes simplex virus (HSV) at a M.O.I.
of S and total RNA was extracted (Chomczynski and Sacchi,
162 Anal. Biochem. 156, 1987). An RNase protection assay
was used to detect mRNA after incubation of total infected
cellular RNA in cytoplasmic extracts. RNA probes were
produced from PCR-amplified template DNA uslng T7 RNA
polymerase (U.S. Biochemical) in the presence of 32p ~-CTP
(3,000;~ C1~/mmole, New England Nuclear, Boston, MA).
~ ~ 20 TempLate~DNA was~ inactivated with 1 unit of RNase-free
;-~ DNaseI for 15 minutes at 37C. Unincorporated nucleotides
were removed by G-50 spin chromatography. Samples (6 ~
were taken from the nuclease assays after various periods
;~ ~of incubation at 37C, added to 40 ~l of 4 M GUSCN buffer
2S (4 M guanidinium thiocyanate; 25 mM sodium citrate, pH 7;
~-; 0.5% sarcosyl; and 0.1 M 2-mercaptoethanol), and 5 ~1 of
32P-labeled RNA probe (5xlOs cpm/5 ~l, specific activity of
1.8x106 cpm/~g) in 4 M GUSCN buffer. Hybridization
reactions were~coveredl with~mineral~oil and lncubated at
55C for 12-16 hours, after which the hybridization
- reaction was mixed with 500 ~l of RNase buffer (0.4 M
NaCl, 20 ~g/ml RNaseA, 2 units~ml T1 RNase) and incubated
for 30 minutes at 37C. RNase activity was quenched by
incubation with 10 ~l of 20% SDS and 10 ~l of proteinase
K (20 mg/ml), and the RNA was extracted using a
phenol/chloroform mixture. The protected RNA fragment was
purified by precipitation with an equal volume of
-~093/23057 ~ 3; ~ ~ ~ PCT/US93/04573
isopropanol in the presence of 20 ~g of carrier yeast
tRNA. The RNA pellets were resuspended in gel loading
buffer, heated to 95C for 5 minutes and separated by
electrophoresis on a 5% polyacrylamide, 7 M urea gel.
Protected fragments were visualized by autoradiography,
and the films were scanned with a sio 5000 density
scanner.
In experiments using these methods, the rate of
digestion of ribozymes in Vero cell extracts was similar,
demonstrating the lack of significant phosphatase activity
in Vero cellular fractions. Similar results were observed
with HeLa cellular fractions. In most extracts, ladders
of digested fragments were observed; such ladders would
not be expected if digestion was an artifact of
phosphatase action. Thus, digestion using 5~ end-labeled
ribozymes is an accurate assessment of nuclease action in
ce~llular extracts.
In other experiments, labeled ribozymes were
..~
incubated in various Vero and HeLa cellular fractions.
Incubation of ribozymes in either membranous or nuclear
~ fractions resulted in a linear decrease of intact
-~ mo-lecules over time. In contrast, no digestion of
~; rlbozymes occurred durin~ a 24 hour incubation in Vero
cytoplasmic extracts, and HeLa cytoplasmic extracts
- 2~ exhibited a 20-30 minute delay in the onset of RNA
digestion. After this refractory period, the rate of
digestion was linear but not as rapid as the rates
observed in any of the nuclear or membranous fractions.
~ ,The effect jofif,our dlvalent cations (Mg~2, Mn~2,
Ca~2, and Zn~2) on the nuclease activity of the cellular
fractions was assessed. Vero cytoplasmic extracts were
~ stimulated by the addition of 1 mM Mg'2 or Mn~2, while Ca~-
-~ or Zn~2 had no effect. Nuclease activity in HeLa
cytoplasmic extracts was enhanced only by the addition of
1 mM Zn~2. Both Vero and HeLa membranous fractions
exhibited maximum nuclease activity with the addition of
Mgt~ or Mn~- ions, while the addition of Ca~- significantly
~ .
:' .
WO93/230~;7 lc ~ PCl~/US93/1)4s7 ~`
38
reduced activity of the HeLa membranous fraction and
abolished nuclease activity in the Vero membranous
fraction. Addition of Zn~2 to both membranous fractions
resulted in a loss of all RNase activity. The Vero
nuclear extract demonstrated roughly equivalent nuclease
activity in the presence of either Mg~- alone or a Mgt2 and
Mn~2 ion combination, less in the presence of Mg2 and Cat2,
and no activity in the presence of Mgt2 and Zn~2. The
effects of cation addition were not as dramatic with HeLa
- 10 nuclear extracts. The nuclease activity of these
fractions was greatest in the presence of Mg~2 alone or Mg~
and Ca~- and decreased slightly with the addition of Mn-- or
Zn~2 to the Mgt~ present in the extracts.
To verify that nuclease activity was dependent ;
lS upon added divalent cations, nuclease assays were
performed uslng 1 mM Mg~2 in the presence and absence of
20 mM EDTA. For the HeLa cytoplasmic fractions,-the Mg~2
was r~eplaced with 1 mM Zn'2. The presence of 20 mM EDTA
; c~omplet~e1y;~abolished nuclease activity in the Vero and
20~HeLa cytoplasmic fractions and Vero nuclear fractions.
~-1 Nuclease acti~ity in the HeLa membranous and nuclear
fractions was partially inhibited by the addition of EDTA,
~ ~ while EDTA had no effect on the nuclease activity in the
-~ - Vero membranous fraction. For comparative purposes,
f~ 25~ reactlons using DNA oligonucleotides were performed using
different Vero fractions. All DNase activity in Vero
~;~ cytoplasmic, membranous, and nuclear fractions was
inhibited by 20 mM EDTA.
The stability of,RNA oligQnucleotides and HSV-1
mRNA were compared in the presence and absence o
activity-enhancing divalent cations (1 mM Mgt2, Vero cells;
-~ ~ 1 mM Zn~2, HeLa cells). Total cellular RNA from HSV-1 ,
~1 infected Vero cells (B mg) and tracer amounts of 32p_5'_ I
;~ end-labeled RNA oligonucleotides (1 pmole) were incubated
with Vero or HeLa cytoplasmic extracts. In the absence of
divalent cations, no substantial decrease of intact
ribozymes was detected in assays, although mRNA was
,:-
~r093/23057 ~1 3X l.'1.'3 PCrlUS93/04~73
39
digested in both vero and HeLa cytoplasmic extracts.After addition of divalent cations, digestion of ribozymes
occurred in both vero and HeLa cytoplasmic fractions. The
rate of ribozyme digestion in HeLa extracts increased to
levels similar to those observed with mRNA, while the rate
of mRNA digestion remained greater than the rate of
ribozyme digestion in Vero cytoplasmic fractions.
Thus, the stability of hammerhead ribozymes were
compared in both Vero and HeLa cell cytoplasmic,
membranous and nuclear fractions. Vero cytoplasmic and
nuclear fractions were found to require Mgt2 for optimal
nuclease activity, while the membranous fraction was no,
altered by the addition of divalent cations. HeLa
membranous and nuclear fractions were also activated by
Mg~2, while the cytoplasmic fractions required Znt2 for
nuclease activation. Relative stabilities of ribozymes
and mRNAs were compared in Vero and HeLa cytoplasmic
fractions. In the absence of appropriate divalent
cations, little ribozyme digestion was observed in either
cytoplasmic preparation while mRNA was rapidly digested.
The addition of Mg~2 to Vero cytoplasmic extracts and Zn~
to the HeLa cytoplasmic extracts stimulated ribozyme
- ~ ~ degradation and enhanced mRNA digestion. These data show
that the nuclease sensitivity of ribozymes is cell-type
specific, varies with the intracellular compartment
; studied and may not be able to be predicted from studies
; with mRNA. Notably, however, ribozymes appear stable in
such cellular fractions for a period of time po~entially
sufficient to haveja ther~peutically useful activity.
Administration of Rlboz~me
Selected ribozymes can be administered
prophylactically, or to patients expressing mdr-1 mRNA, or
~ ha~ing CM1, leukemic conditions, Burkitt's lymphoma,
-~ follicular lymphoma, breast cancer, colon carcinoma,
~- 35 neuroblastoma, lung cancer, or pretumor cells, e.q., b~-
exogenous deli~ery of the ribozyme to an a desired tissue
by means of an appropriate delivery vehicle, e.a., a
W093/~30s7 ~ PCT/US93/0457,
liposome, a controlled release vehicle, by use of
iontophoresis, electroporation or ion paired molecules, or
covalently attached adducts, and other pharmacologically
approved methods of delivery. Routes of administration
include intramuscular, aerosol, oral (tablet or pill
form), topical, systemic, ocular, intraperitoneal and/or
intrathecal. Alternatively, ribozymes may be administered
to a tissue or afflicted cell ex vivo to eradicate
tumorigenic cells prior to re-implantation (e.a., in the
course of autologous bone marrow transplantation therapy).
Expression vectors for immunization with ribozymes and/or
delivery of ribozymes are also suitable.
The specific delivery route of any selected
ribozymie will depend on the use of the ribozyme.
Generally, a specific delivery program for each ribozyme
; will focus on unmodified ribozyme uptake with regard to
intracellular localization, followed by demonstration of
efficacy. ~Alternatively, delivery to these same cells in
an organ or tissue of an animal can be pursued. Uptake
~stud1es will-include uptake assays to evaluate cellular
ribozyme uptake, regardless of the delivery vehicle or
strategy. Such assays will also determine the
intracellular localization o`f the ribozyme following
uptake, ultimately establishing the requirements for
malntenance of steady-state concentrations within the
cellular ~ompartment containing the target sequence
- (nucleus and/or cytoplasm). Efficacy and cytotoxicity can
then be tested. Toxicity will not only include cell
viability but also cell function.
" ~
Some methods of delivery that may be used
include:
a. encapsulation in liposomes,
b. transduction by retroviral vectors,
c. conjugation with cholesterol,
d. localization to nuclear compartment
utilizing nuclear targeting site found on
most nuclear proteins,
v~O 93/23057 ~ l 3 ~ PCT/US93/04573
41
e. neutralization of charge of ribozyme by
using nucleotide derivatives,
f. use of blood stem cells to distribute
ribozymes throughout the body, and
g. electroporation~
At least three types of delivery strategies are
useful in the present invention, including- ribozyme
modifications, particle carrier drug delivery vehicles,
and retroviral expression vectors. Unmodified ribozvmes,
10 like most small molecules, are taken up by cells, albeit ;
slowly. To enhance cellular uptake, the ribozyme may be
modified essentially at random, in ways which reduce its
charge but maintains specific functional groups. This
results in a molecule which is able to diffuse across the
cell membrane, thus removing the permeability barrier.
Modification of ribozymes to reduce charge is
just one approach to enhance the cellular uptake of these
larger~molecules. The random approach, however, is not
~- ~ advisable since ribozymes are structurally and
~functionally more complex than small drug molecules. The
structural requirements necessary to maintain ribozyme
catalytic activity are well understood by those in the
- art. These requirements are taken into consideration when
~;~ designing modifications to enhance celIular delivery. The
- ~ 25 modifications are also designed to reduce susceptibility
to nuclease degradation. Both of these characteristics
should greatly improve the efficacy of the ribozyme.
~ellular uptake can be increased by several orders of
~magnitude without having to alter the phosphodiester
linkages necessary for ribozyme cleavage activity.
Chemical modifications of the phosphate b~ckbone
;~ will reduce the negative charge allowing free diffusion
across the membrane. This principle has been successfully
demonstrated for antisense DNA technology. The
similarities in chemical composition between DNA and RNA
make this a feasible approach. In the body, maintenance
of an external concentration will be necessary to drive
W093/23057 ~1 3~ ~ ~r! ~; PCT/US93/045~
e: cJ ~
42
the diffusion of the modified ribo~yme into the cells of
the tissue. Administration routes which allow the
diseased tissue to be exposed to a transient high
concentration of the drug, which is slowly dissipated by
systemic adsorption are preferred. Intravenous
administration with a drug carrier designed to increase
the circulation half-life of the ribozyme can be used.
The size and composition of the drug carrier restricts
rapid clearance from the blood stream. The carrier, made
to accumulate at the site of infection, can protect the
-ribozyme from degradative processes.
Drug delivery vehicles are effective for both
systemic and topical administration. They can be designed
to serve as a slow release reservoir, or to deliver their
contents directly to the target cell. An advantage of
using ~direct delivery drug vehicles is that multiple
molecules are delivered per uptake. Such vehicles have
beén~shown to increase the circulation half-life of drugs
whlch~would~o~therwise~be rapidly cleared from the blood
stream~ Some~examples of such specialized drug delivery
vehicles ~whlch fall into this category ar~e liposomes,
hyd~rogels, cyclodextrins, biodegradable nanocapsules, and
bloadhesive microspheres.
From this category of delivery systems,
liposomes are preferred. Liposomes increase intracellular
;stabil~ity, increase uptake efficiency and improve
biological activity.
Liposomes are hollow spherical vesicles composed
of lipids arranged in a similar fashion as those lipids
which make up the cell membrane. They have an internal
, - :
aqueous space for entrapping water soluble compounds and
-~ range in size from 0.05 to several microns in diameter.
~-~ Several studies have shown that liposomes can deliver RNA
to cells and that the RNA remains biologically active.
35For example, a liposome delivery vehicle
;; originally designed as a research tool, Lipofectin, has
~ - 3 ' ~ ~
.j~' ~',' .
~,.''~,:
`~093/23057 ;~ PCT/US93/04573
! ~
43
been shown to deliver intact mRNA molecules to cells
yielding production of the corresponding protein.
Liposomes offer several advantages: They are
non-toxic and biodegradable in composition, they display
long circulation hal~-lives; and recognition molecules can
be readily attached to their surface for targeting to
tissues. Finally, cost effective manufacture of liposome-
based pharmaceuticals, either in a liquid suspension or
lyophilized product, has demonstrated the viability of
this technology as an acceptable drug delivery system.
Other controlled release drug delivery systems,
such as nonoparticles and hydrogels may be potential
delivery vehicles for a ribozyme. These carriers have
been developed for chemotherapeutic agents and protein-
based pharmaceuticals, and consequently, can be adaptedfor ribozyme delivery.
;~ ~ Topical adm1nistration of ribozymes is
ad~antageous since it allows localized concentration at
` the slte of administration with minimal systemic
adsorption.~ This simplifies the delivery strategy of the
ribozyme~to the disease site and reduces the extent of
toxicological characterization. Furthermore, the amount
of mat~erlal to be applied is far less than that required
- for other administration routes. Effective delivery
25 ~requires the ribozyme to diffuse into the infected cells
-or through the skin to the underlying vasculature.
Chemical modification of the ribozyme to neutralize
negative charge may be all that is required for
penetration. However, in the event that charge
neutralization is insufficient, the modified'ribozyme can
be co-formulated with permeability enhancers, such as
Azone or oleic acid, in a liposome. The liposomes can
.,, ~, ,.
either represent a slow release presentation vehicle in
which the modified ribozyme and permeability enhancer
transfer from the liposome into the infected cell, or the
liposome phospholipids can participate directly with the
modified ribozyme and permeability enhancer in
~' ~
, - .
W093/23057 PCT/US93/045
44
facilitating cellular delivery. In some cases, both the
ribozyme and permeability enhancer can be formulated into
a suppository formulation for slow release.
Ribozymes may also be systemically administered.
Systemic absorption refers to the accumulation of drugs in
the blood stream followed by distribution throughout the
entire body. Administration routes which lead to systemic
absorption include: intxavenous, subcutaneous,
intraperitoneal, intranasal, intrathecal and ophthalmic.
Each of these administration routes expose the ribozyme to
an accessible diseased tissue. Subcutaneous
administration drains into a localized lymph node which
proceeds through the lymphatic network into the
circulation. The rate of entry into the circulation has
been shown to be a function of molecular weight or size.
The use of a liposome or other drug carrier localizes the
ribozyme at the lymph node. The ribozyme can be modified
to diffuse into the cell, or the liposome can directly
participate in the delivery of either the unmodified or
modified ribozyme to the cell.
A liposome formulation containing phosphatidyl-
ethanolomidomethylthiosuccinimide which can deliver
oligonucleotides to lymphocytes and macrophages is also
useful for certain cancerous conditions. Furthermore, a
200 ~m diameter liposome of this composition was
internalized as well as 100 nm diameter liposomes. The
200 nm liposomes exhibit a 10-fold greater packaging
capacity than the 100 nm liposomes and can accomodate
larger molecules such as a ribozyme expression vector.
This oligonucleotide delivery system inhibits viral
proliferation in these viruses that infect primary immune
cells. This oligonucleotide delivery system prevents mRNA
expression in affected primary immune cells. Whole blood
studies show that the formulation is taken up by 90% of 35 the lymphocytes after 8 hours at 37C. Preliminary
biodistribution and pharmacokinetic studies yielded 70~ of
~093J23057 PCT/US93/04573 ~
,
the injected dose/gm of tissue in the spleen after 1 hour
following intravenous administration.
Intraperitoneal administration also leads to
entry into the circulation with the molecular weight or
size controlling the rate of entry.
Liposomes injected intravenously show
accumulation in the liver, lung and spleen. The
- composition and size can be adjusted so that this
accumulation represents 30% to 40% of the injected dose.
The remaining dose circulates in the blood stream for up
t o 24 hours. `
The chosen method of delivery should result in
cytoplasmic accumulation and molecules should have some
nuclease-resistance for optimal dosing. Nuclear delivery
may be used but is less preferable. Most preferred
deliver~y~methods include liposomes (10-400 nm), hydrogels,
controlled-release polymers, microinjection or
electroporation~ (for ex vivo treatments) and other
- pharmaceutically applicable vehicles. The dosage will
20' depend upon the disease indication and the route of
; admin1stratlon but should be between 100-200 mg/kg of body
weight~/day. The duration of treatmen~ will extend through
the course of the disease symptoms, possibly continuously.
-~ The number of~ doses will depend `upon disease delivery
-25 vehicle and efficacy data from clinical trials. ;`
Est~ablishment of therapeutic levels of ribozyme
within~the cell lS dependent upon the rate of uptake and
degradation. Decreasing the dègree of degradation will
prolong the intfracellular; half-life of the ribozyme.
Thus, chemically modified ribozymes, e.a., with
modification of the phosphate backbone, or capping of the
5' and 3' ends of the rlbozyme with nucleotide analogs may
require different dosaging. Descriptions of useful
systems are provided in the art cited above, all of which
is hereby incorporated by reference herein.
The claimed ribozymes are also useful as
diagnostic tools to specifically or non-specifically
~ .
W093/23057 PCT/US93/0457'~-
..
46
detect the presence of a target RNA in a sample. That is,
the target RNA, if present in the sample, will be
specifically cleaved by the ribozyme, and thus can be
readily and specifically detected as smaller RNA species.
S The presence of such smaller RNA species is indicative of
the presence of the target RNA in the sample.
Other embodiments are within the following
claims.
.
.
